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bull Cancer is the second leading cause
of death worldwide
Despite some successful intervention
strategies in reduction of cancer-related
mortality cancer is still considered a
deadly disease
It is estimated that about one and half
million new cases and half million
deaths from cancer occur in the United
States in 2009 Jemal A Siegel R Ward E Hao Y Xu J Thun MJ
Cancer statistics 2009 CA Cancer J Clin 200959225-49
To overcome cancer mortality the current medical
focus has been centered on the development of novel
biomarkers that can be used for diagnosis prognosis or
therapy of cancer
The immune response targets are revealed to be
promising cancer biomarkers
Cancer patients produce
autoantibodies to proteins
that are mutated misfolded
improperly post-translationally
modified overexpressed
truncated or aberrantly localized
in tumor cells
A large number of tumor antigens that elicit autoantibodies have been identified by different methods
Breast cancer is a major cancer cause and death worldwide
Several breast cancer biomarkers such as BRCA estrogen
receptor and HER2 have been shown to have a diagnostic
prognostic or therapeutic value
However they are reliable just in a proportion of patients and
search for finding novel breast cancer biomarkers is underway
The aim of our study was to identify antigens eliciting a humoral
immune response in HER2+ and HER2 NP breast cancers by
two dimensional electrophoresis (2D) Western blotting and
mass spectrometry
Serum samples Our study groups comprised of 18 breast cancer patients
and 9 normal individuals Breast cancer patients had at least one involved
auxiliary lymph node 9 were HER2 negative and 9 were HER2 positive (3+)
Normal individuals had no history of malignancy
autoimmune disease or severe inflammatory disease
The MCF7 (an ER+ low-expressing HER2 cell line) and SKBR3(an ER- high expressing
HER2 cell line) breast cancer cell lines cultured in culture media contained RPMI 1640
medium 10 fetal bovine serum 100UmL penicillin and 100 mgmL streptomycin
Breast cancer cell line was lysed in a lysis solution (7 M Urea 2 M Thoiurea CHAPS 4 DTT
50 mM IPG buffer 1)
Cell lysates were subjected to 2D-PAGE
2D gels were either stained with blue silver method (Staining solution Coomassie Brilliant
blue MeOH orthophosphoric acid ammonium sulfate) or transferred to PVDF membranes
Immuno-reactive protein spots were punched from the gel
and transferred to a 96 well polypropylene plate
Spots were sent for matrix-assisted laser desorptionionisation time-
of-flighttime-of-flight(MALDI-TOFTOF)mass spectrometry
analysis
2 Dimensional Electrophoresis
Proteomics is the large-scale study of proteins
particularly their structures and functions
The proteome is the entire complement of proteins produced by an organism or system This will vary with time
Proteomics is often considered the next step in the study of biological systems after genomics
Why study Proteomics
First the level of transcription of a gene gives only a rough estimate of its level of
expression into a protein An mRNA produced in abundance may be degraded rapidly
or translated inefficiently resulting in a small amount of protein
Second many proteins experience post-translational modifications that profoundly
affect their activities for example some proteins are not active until they become
phosphorylated Methods such as phosphoproteomics and glycoproteomics are used to
study post-translational modifications
Third many transcripts give rise to more than one protein through alternative splicing
or alternative post-translational modifications
Finally At present there is no other technique that is capable of simultaneously
resolving thousands of proteins in one separation procedure
The core technology of proteomics is 2-DE
To identify as many components of the proteome as possible
Protein Mining catalog all the proteins present in a tissue cell organelle etc
Protein Function
Mapping Protein Post-Translational Modification - Characterization of posttranslational modifications phosphorylation glycosylation oxidation etc
Protein Localization
Protein Expression Studies
Comparative proteomics
- proteomics monitors the expression of a large number of proteins within a cell or tissue and observers quantitatively how the
patte of expression changes under different circumstances (comparative proteomics) such as
bull Healthy Diseased
bull Cancerous Benign
bull Drug resistant Drug susceptible
bull Tissue specific
An interesting use of proteomics is using specific protein biomarkers to diagnose disease
Sample Preparation 2-D Electrophoresis Spot Detection amp Image Analysis
Enzymatic Digestion Peptide-Mass Fingerprinting
Peptide Sequencing via MS Database Search
Protein Identification
(I) (III)
(IV) (V) (VI)
(II)
Sample preparation buffer composition IPG strip rehydration 1st dimension IEF run IPG strip wash Equilibration buffer Equilibration buffer 2nd dimension SDS-PAGE Gel staining Immunoblot Imaging
Sample preparation
buffer composition
IPG strip rehydration
1st dimension IEF run
IPG strip wash
Equilibration buffer I
Equilibration buffer II
2nd dimension SDS-PAGE
Gel staining
Imaging
2-D Electrophoresis
Consistent protocol Limit sample degradation
bull fresh cellstissue bull protease inhibitors bull keep sample cold bull long term storage at ndash80degC
Limit sample contamination
bull gloves (keratin)
SolubilisationDenaturation buffer
bull separate proteins into individual components
bull reliable running in the IEF
Gentle lysis method 1 Osmotic lysis (cultured cells)
Suspend cells in hypoosmotic solution
2 Repeated freezing and thawing (bacteria)
Freeze using liquid nitrogen
3 Detergent lysis (yeast and fungi)
Lysis buffer (containing urea and detergent)
SDS (have to be removed before IEF)
4 Enzymatic lysis (plant bacteria fungi)
Lysomzyme (bacteria)
Cellulose and pectinase (plant)
Lyticase (yeast)
Vigorous lysis method
1 Sonication probe (cell suspension)
Avoid overheat cool on ice between burst
2 French pressure (microorganism with cell wall)
Cells are lysed by shear force
3 Mortar and pestle (solid tissue microorganism)
Grind solid tissue to fine powder with liquid nitrogen
4 Sample grinding kit (for small amount of sample)
For precious sample
5 Glass bead (cell suspension microorganism)
Using abrasive vortexed bead to break cell walls
Chaotrope
Urea (up to 9 M)
Thiourea (up to 2 M)
bull Disrupt of hydrogen and hydrophobic bonds
Note Urea (if tdeggt37degC) cyanate (HN=C=O)
carbamylation (Lys Arg)
(R-NH2 R-NH-CO-NH2)
carbamylation trains
Reductants
b-mercaptoethanol
DTT(dithiothreitol)
bull Break disulfide bridge
(within or between protein)
Detergents ndash surfactants
CHAPS (up to 4)
bull Disrupt membranes
bull Break hydrophobic interactions
bull Solubilise lipids
bull Release membrane bound proteins
Note No net charge for IEF (no SDS)
Soluble in urea
Isoelectric Focusing
Measure the protein concenteration in you samples
1 Biuret
2 Lowry methods
3 Bradford methods
4 UV methods
5 Special methods
6 Other commercial methods
BCA assay (bicinchoninic acid assay Pierce)
DC protein assay (detergent compatible Bio-rad)
DCRC protein assay (detergentreducing agent compatible
Bio-rad)
Before runninng IEF you shouldhellip
Introduced by Gorg A
Ref Gorg A (1994) Westermeier (2001)
Dried gel strips can be stored at -20 to -
80 from months to years
Amersham Biosciences Bio-Rad
IPG strips (3 mm x 18 cm x 05 mm)
Narrow range
Medium range
Broad range
4 7
35 45 55 67
40 50 60
3 10
6 11
Load 400 ml sample per groove (no bubbles)
Place IPG strip gel facing down in groove
Add ~25 ml non conductive oil per groove
Peel off protective film from strip
Rehydrate overnight (~22 hrs) at room temperature
Program power supply
bull Number of gels (1-10)
bull Max voltage 5000 V
bull Vhold 125 V
bull Duration 24 hrs 00 min
bull Max current 80 mAstrip
bull Volt hours 80000 Vh
pI
+
+ - - - - - -
+ + + + + + + -
pH 4 pH 7
Cathode Anode
Place wet wicks (DH2O) under each end of strip
Set chiller temperature for 20degC (setting ~25)
Remove IPG strip from tray
Let oil drip off the strip
Place IPG strip gel facing up in equilibration tray
+ -
Add 10 ml equilibration buffer 1 per tray
+ -
+ -
+ -
+ -
(in chemical hood)
MW 4 7
+ -
10 DuracrylTM gel (22 cm x 23 cm x 1 mm)
bull TrisTricineSDS buffer
bull IPG strip
bull MW (gel worm)
bull Agarose overlay
bull Silicone spacer
bull Gasket
-
+
Conditions
bull Number of gel (1-10)
bull Max voltage 500 V
bull Max power 1600 mWgel
bull 4degC (setting 10)
bull ~19 hrs
10 DuracrylTM gel (22 cm x 23 cm x 1 mm)
MW 4 7
+ -
Trisacetate 25 mM Tris-base pH 83 (acetic acid)
200 mM Tris-base 200 mM Tricine 04 SDS
045 mm filtered
TrisTricine SDS
Bio-Rad
Digest to peptide fragment MS analysis
1 First dimension
isoelectric focusing
separation according to the pI
2 Second dimension
SDS electrophoresis (SDS-PAGE)
Separation according to the MW
Interested spot
Sensitivity Process
TimeSteps
Advantages
bullBio-safe Coomassie 10ng 25hr3 steps MS compatible easily visualised non
hazardous
bullCoomassie Blue R-250 40ng 25hr2 steps Oldest and least expensive method
bullSilver Stain Plus 1ng 15hr3 steps MS compatible high sensitivity low
background
bullBio-Rad silver stain 1ng 2hr7 steps High sensitivity detects some highly
glycosylated and other difficult to
stain proteins
bullSypro ruby 1ng 3hr2 steps MS compatible allows analysis in
flourescent imagers linear over 3
orders of magnitude
Coomassie staining
bull moderate sensitivity (36-47 ng)
bull non specific
bull not quantitative
Silver staining
bull sensitive (05-12 ng)
bull time consuming
bull non specific
bull negative stain some spots
Fluorescent dye (SYPRO ruby)
bull sensitive (1-2 ng)
bull specific quantitative
bull end point stain
Coomassie Silver
Coomassie Silver Stain Copper Stain
SDS-polyacrylamide gel
Electrotransfer to membrane
Phosphoimager for 32P and 35S labelled
1D or 2D gels
Fluoroimager for
SYPRO labelled
1D or 2D gels
Densitometer or
Photo Scanner
Normal liver
Tumor
Both
Melanie 4 (GeneBio - Windows only)
httpcaexpasyorgmelanie
ImageMaster 2D Elite (Amersham)
httpwwwimsupportcom
Phoretix 2D Advanced
httpwwwphoretixcom
PDQuest 61 (BioRad - Windows only) httpwwwproteomeworksbio-radcomhtmlpdquesthtml
Mass Spectrometry
wwwexpasych - Swiss-2DPAGE httpwwwanlgovBIOPMG - Mouse liver human breast cell lines pyrococcus Argonne Protein Mapping Group httpwwwharefieldnthamesnhsuknhliproteinindexhtml - HSC-2DPAGE Heart Science Centre Harefield Hospital httpotowustleduthcperi-gelshtm - Washington Univ Inner Ear Protein Database
httpcaexpasyorgch2d2d-indexhtml - World 2DPAGE Index of 2D gel databases
Time line ~ 4 days
bull Protein gt 250 kDa do not enter 2 SDS-PAGE properly
bull PH more than 9
bull PH less than 4
bull Hydrophobic proteins
bull Low abundant proteins
Immunoblotting Proteins from 2D gels were transferred to PVDFmembranes The membranes were blocked with 5 skimmed milk in PBS-Tween (PBS-T) for 3 h The membranes were incubated overnight at 4degC with individual sera diluted 1100 in PBS-T followed by secondary antibody (anti-human IgG conjugated with horseradish peroxidase) for 2 h with dilutions of 13000 Before and after addition of the secondary antibody membranes washed 4 times each time for 5 min in PBS-T To visualize antigenic components membranes were incubated in diaminobenzeden (DAB) and H2O2 for 15 min
List of protein spots that reacted with more than 30 of serum samples
and were identified by mass spectrometry
1Heat shock protein HSP 90-beta
2 precursor(GRP 78) 78 kDa glucose-regulated protein
3 Heat shock cognate 71 kDa protein
4 Stress-70 protein mitochondrial precursor
5 Tubulin beta-5 chain
6Tubulin alpha-6 chain and 60 kDa heat shock protein mitochondrial
precursor (Hsp60)
7 Heterogeneous nuclear ribonucleoprotein K (hnRNP K)
8 Cytokeratin 8
9 Cytokeratin 18
10 Beta-actin
11 cytokeratin 19
12 Eukaryotic initiation factor 4A-I
13 14-3-3 protein epsilon
14 14-3-3 protein zetadelta (Protein kinase Cinhibitor protein-1)
15 Translationally controlled tumor protein (TCTP)
Among these autoantigens cytokeratin 8 cytokeratin 18 Fructose-bisphosphate aldolase and enolase were reacted with more than 60 of healthy individualsrsquo serum samples These antigens were intense spots in gels
Heat Shock proteins tubulins heterogeneous nuclear ribonucleoprotein actins cytokeratins 14-3-3 protein epsilon 14-3-3 protein zetadelta eukaryotic initiation factors Rho GDP-dissociation inhibitors phosphoglycerate mutase 1 G6PD GA3PD peroxiredoxins F-acting capping protein and triosephosphate isomerase were previously reported to induce humoral arm of the immune response in cancer
Translationally controlled tumor protein and electron transfer flavoprotein alpha-subunit have not been reported to induce humoral arm of the immune response in cancer so far
In terms of biological processes the identified proteins
belong to several groups
They are mainely involved in cell motility cytoskleton
stress response apoptosis cell signaling and
metabolism
Identification of other immunogenic proteins is
underway
Appel RD et al 1993 - SWISS-2DPAGE a database of two-dimensional gel electrophoresis images Electrophoresis 14 1232-1238
Appel RD Bairoch A Sanchez JC Vargas JR Golaz O Pasquali C and Hochstrasser DF 1996 ndash Federated two-dimensional electrophoresis database a simple means of publishing two-dimensional electrophoresis data Electrophoresis 17 540-546
Bjellqvist B Ek K Righetti PG Gianazza E Gorg A Westermeier R Postel W 1982 ndash Isoelectric focusing in immobilised pH gradients principle methodology and applications JBiochemBiophysMethods 6 317-339
Brazma A et al 2001 ndash Minimum information about a microarray experiment (MIAME)-towards standards for microarray data Nat genetics 29 365-71
Hoogland C Baujard Sanchez JC Hochstrasser DF and Appel RD 1997 ndash Make2ddb a simple package to set up a two-diensional electrophoresis database for the world wide web Electrophoresis 18 2755-2758
OFarrell 1975 - High resolution two-dimensional electrophoresis of proteins JBiolChem 25 250 4007-21
To overcome cancer mortality the current medical
focus has been centered on the development of novel
biomarkers that can be used for diagnosis prognosis or
therapy of cancer
The immune response targets are revealed to be
promising cancer biomarkers
Cancer patients produce
autoantibodies to proteins
that are mutated misfolded
improperly post-translationally
modified overexpressed
truncated or aberrantly localized
in tumor cells
A large number of tumor antigens that elicit autoantibodies have been identified by different methods
Breast cancer is a major cancer cause and death worldwide
Several breast cancer biomarkers such as BRCA estrogen
receptor and HER2 have been shown to have a diagnostic
prognostic or therapeutic value
However they are reliable just in a proportion of patients and
search for finding novel breast cancer biomarkers is underway
The aim of our study was to identify antigens eliciting a humoral
immune response in HER2+ and HER2 NP breast cancers by
two dimensional electrophoresis (2D) Western blotting and
mass spectrometry
Serum samples Our study groups comprised of 18 breast cancer patients
and 9 normal individuals Breast cancer patients had at least one involved
auxiliary lymph node 9 were HER2 negative and 9 were HER2 positive (3+)
Normal individuals had no history of malignancy
autoimmune disease or severe inflammatory disease
The MCF7 (an ER+ low-expressing HER2 cell line) and SKBR3(an ER- high expressing
HER2 cell line) breast cancer cell lines cultured in culture media contained RPMI 1640
medium 10 fetal bovine serum 100UmL penicillin and 100 mgmL streptomycin
Breast cancer cell line was lysed in a lysis solution (7 M Urea 2 M Thoiurea CHAPS 4 DTT
50 mM IPG buffer 1)
Cell lysates were subjected to 2D-PAGE
2D gels were either stained with blue silver method (Staining solution Coomassie Brilliant
blue MeOH orthophosphoric acid ammonium sulfate) or transferred to PVDF membranes
Immuno-reactive protein spots were punched from the gel
and transferred to a 96 well polypropylene plate
Spots were sent for matrix-assisted laser desorptionionisation time-
of-flighttime-of-flight(MALDI-TOFTOF)mass spectrometry
analysis
2 Dimensional Electrophoresis
Proteomics is the large-scale study of proteins
particularly their structures and functions
The proteome is the entire complement of proteins produced by an organism or system This will vary with time
Proteomics is often considered the next step in the study of biological systems after genomics
Why study Proteomics
First the level of transcription of a gene gives only a rough estimate of its level of
expression into a protein An mRNA produced in abundance may be degraded rapidly
or translated inefficiently resulting in a small amount of protein
Second many proteins experience post-translational modifications that profoundly
affect their activities for example some proteins are not active until they become
phosphorylated Methods such as phosphoproteomics and glycoproteomics are used to
study post-translational modifications
Third many transcripts give rise to more than one protein through alternative splicing
or alternative post-translational modifications
Finally At present there is no other technique that is capable of simultaneously
resolving thousands of proteins in one separation procedure
The core technology of proteomics is 2-DE
To identify as many components of the proteome as possible
Protein Mining catalog all the proteins present in a tissue cell organelle etc
Protein Function
Mapping Protein Post-Translational Modification - Characterization of posttranslational modifications phosphorylation glycosylation oxidation etc
Protein Localization
Protein Expression Studies
Comparative proteomics
- proteomics monitors the expression of a large number of proteins within a cell or tissue and observers quantitatively how the
patte of expression changes under different circumstances (comparative proteomics) such as
bull Healthy Diseased
bull Cancerous Benign
bull Drug resistant Drug susceptible
bull Tissue specific
An interesting use of proteomics is using specific protein biomarkers to diagnose disease
Sample Preparation 2-D Electrophoresis Spot Detection amp Image Analysis
Enzymatic Digestion Peptide-Mass Fingerprinting
Peptide Sequencing via MS Database Search
Protein Identification
(I) (III)
(IV) (V) (VI)
(II)
Sample preparation buffer composition IPG strip rehydration 1st dimension IEF run IPG strip wash Equilibration buffer Equilibration buffer 2nd dimension SDS-PAGE Gel staining Immunoblot Imaging
Sample preparation
buffer composition
IPG strip rehydration
1st dimension IEF run
IPG strip wash
Equilibration buffer I
Equilibration buffer II
2nd dimension SDS-PAGE
Gel staining
Imaging
2-D Electrophoresis
Consistent protocol Limit sample degradation
bull fresh cellstissue bull protease inhibitors bull keep sample cold bull long term storage at ndash80degC
Limit sample contamination
bull gloves (keratin)
SolubilisationDenaturation buffer
bull separate proteins into individual components
bull reliable running in the IEF
Gentle lysis method 1 Osmotic lysis (cultured cells)
Suspend cells in hypoosmotic solution
2 Repeated freezing and thawing (bacteria)
Freeze using liquid nitrogen
3 Detergent lysis (yeast and fungi)
Lysis buffer (containing urea and detergent)
SDS (have to be removed before IEF)
4 Enzymatic lysis (plant bacteria fungi)
Lysomzyme (bacteria)
Cellulose and pectinase (plant)
Lyticase (yeast)
Vigorous lysis method
1 Sonication probe (cell suspension)
Avoid overheat cool on ice between burst
2 French pressure (microorganism with cell wall)
Cells are lysed by shear force
3 Mortar and pestle (solid tissue microorganism)
Grind solid tissue to fine powder with liquid nitrogen
4 Sample grinding kit (for small amount of sample)
For precious sample
5 Glass bead (cell suspension microorganism)
Using abrasive vortexed bead to break cell walls
Chaotrope
Urea (up to 9 M)
Thiourea (up to 2 M)
bull Disrupt of hydrogen and hydrophobic bonds
Note Urea (if tdeggt37degC) cyanate (HN=C=O)
carbamylation (Lys Arg)
(R-NH2 R-NH-CO-NH2)
carbamylation trains
Reductants
b-mercaptoethanol
DTT(dithiothreitol)
bull Break disulfide bridge
(within or between protein)
Detergents ndash surfactants
CHAPS (up to 4)
bull Disrupt membranes
bull Break hydrophobic interactions
bull Solubilise lipids
bull Release membrane bound proteins
Note No net charge for IEF (no SDS)
Soluble in urea
Isoelectric Focusing
Measure the protein concenteration in you samples
1 Biuret
2 Lowry methods
3 Bradford methods
4 UV methods
5 Special methods
6 Other commercial methods
BCA assay (bicinchoninic acid assay Pierce)
DC protein assay (detergent compatible Bio-rad)
DCRC protein assay (detergentreducing agent compatible
Bio-rad)
Before runninng IEF you shouldhellip
Introduced by Gorg A
Ref Gorg A (1994) Westermeier (2001)
Dried gel strips can be stored at -20 to -
80 from months to years
Amersham Biosciences Bio-Rad
IPG strips (3 mm x 18 cm x 05 mm)
Narrow range
Medium range
Broad range
4 7
35 45 55 67
40 50 60
3 10
6 11
Load 400 ml sample per groove (no bubbles)
Place IPG strip gel facing down in groove
Add ~25 ml non conductive oil per groove
Peel off protective film from strip
Rehydrate overnight (~22 hrs) at room temperature
Program power supply
bull Number of gels (1-10)
bull Max voltage 5000 V
bull Vhold 125 V
bull Duration 24 hrs 00 min
bull Max current 80 mAstrip
bull Volt hours 80000 Vh
pI
+
+ - - - - - -
+ + + + + + + -
pH 4 pH 7
Cathode Anode
Place wet wicks (DH2O) under each end of strip
Set chiller temperature for 20degC (setting ~25)
Remove IPG strip from tray
Let oil drip off the strip
Place IPG strip gel facing up in equilibration tray
+ -
Add 10 ml equilibration buffer 1 per tray
+ -
+ -
+ -
+ -
(in chemical hood)
MW 4 7
+ -
10 DuracrylTM gel (22 cm x 23 cm x 1 mm)
bull TrisTricineSDS buffer
bull IPG strip
bull MW (gel worm)
bull Agarose overlay
bull Silicone spacer
bull Gasket
-
+
Conditions
bull Number of gel (1-10)
bull Max voltage 500 V
bull Max power 1600 mWgel
bull 4degC (setting 10)
bull ~19 hrs
10 DuracrylTM gel (22 cm x 23 cm x 1 mm)
MW 4 7
+ -
Trisacetate 25 mM Tris-base pH 83 (acetic acid)
200 mM Tris-base 200 mM Tricine 04 SDS
045 mm filtered
TrisTricine SDS
Bio-Rad
Digest to peptide fragment MS analysis
1 First dimension
isoelectric focusing
separation according to the pI
2 Second dimension
SDS electrophoresis (SDS-PAGE)
Separation according to the MW
Interested spot
Sensitivity Process
TimeSteps
Advantages
bullBio-safe Coomassie 10ng 25hr3 steps MS compatible easily visualised non
hazardous
bullCoomassie Blue R-250 40ng 25hr2 steps Oldest and least expensive method
bullSilver Stain Plus 1ng 15hr3 steps MS compatible high sensitivity low
background
bullBio-Rad silver stain 1ng 2hr7 steps High sensitivity detects some highly
glycosylated and other difficult to
stain proteins
bullSypro ruby 1ng 3hr2 steps MS compatible allows analysis in
flourescent imagers linear over 3
orders of magnitude
Coomassie staining
bull moderate sensitivity (36-47 ng)
bull non specific
bull not quantitative
Silver staining
bull sensitive (05-12 ng)
bull time consuming
bull non specific
bull negative stain some spots
Fluorescent dye (SYPRO ruby)
bull sensitive (1-2 ng)
bull specific quantitative
bull end point stain
Coomassie Silver
Coomassie Silver Stain Copper Stain
SDS-polyacrylamide gel
Electrotransfer to membrane
Phosphoimager for 32P and 35S labelled
1D or 2D gels
Fluoroimager for
SYPRO labelled
1D or 2D gels
Densitometer or
Photo Scanner
Normal liver
Tumor
Both
Melanie 4 (GeneBio - Windows only)
httpcaexpasyorgmelanie
ImageMaster 2D Elite (Amersham)
httpwwwimsupportcom
Phoretix 2D Advanced
httpwwwphoretixcom
PDQuest 61 (BioRad - Windows only) httpwwwproteomeworksbio-radcomhtmlpdquesthtml
Mass Spectrometry
wwwexpasych - Swiss-2DPAGE httpwwwanlgovBIOPMG - Mouse liver human breast cell lines pyrococcus Argonne Protein Mapping Group httpwwwharefieldnthamesnhsuknhliproteinindexhtml - HSC-2DPAGE Heart Science Centre Harefield Hospital httpotowustleduthcperi-gelshtm - Washington Univ Inner Ear Protein Database
httpcaexpasyorgch2d2d-indexhtml - World 2DPAGE Index of 2D gel databases
Time line ~ 4 days
bull Protein gt 250 kDa do not enter 2 SDS-PAGE properly
bull PH more than 9
bull PH less than 4
bull Hydrophobic proteins
bull Low abundant proteins
Immunoblotting Proteins from 2D gels were transferred to PVDFmembranes The membranes were blocked with 5 skimmed milk in PBS-Tween (PBS-T) for 3 h The membranes were incubated overnight at 4degC with individual sera diluted 1100 in PBS-T followed by secondary antibody (anti-human IgG conjugated with horseradish peroxidase) for 2 h with dilutions of 13000 Before and after addition of the secondary antibody membranes washed 4 times each time for 5 min in PBS-T To visualize antigenic components membranes were incubated in diaminobenzeden (DAB) and H2O2 for 15 min
List of protein spots that reacted with more than 30 of serum samples
and were identified by mass spectrometry
1Heat shock protein HSP 90-beta
2 precursor(GRP 78) 78 kDa glucose-regulated protein
3 Heat shock cognate 71 kDa protein
4 Stress-70 protein mitochondrial precursor
5 Tubulin beta-5 chain
6Tubulin alpha-6 chain and 60 kDa heat shock protein mitochondrial
precursor (Hsp60)
7 Heterogeneous nuclear ribonucleoprotein K (hnRNP K)
8 Cytokeratin 8
9 Cytokeratin 18
10 Beta-actin
11 cytokeratin 19
12 Eukaryotic initiation factor 4A-I
13 14-3-3 protein epsilon
14 14-3-3 protein zetadelta (Protein kinase Cinhibitor protein-1)
15 Translationally controlled tumor protein (TCTP)
Among these autoantigens cytokeratin 8 cytokeratin 18 Fructose-bisphosphate aldolase and enolase were reacted with more than 60 of healthy individualsrsquo serum samples These antigens were intense spots in gels
Heat Shock proteins tubulins heterogeneous nuclear ribonucleoprotein actins cytokeratins 14-3-3 protein epsilon 14-3-3 protein zetadelta eukaryotic initiation factors Rho GDP-dissociation inhibitors phosphoglycerate mutase 1 G6PD GA3PD peroxiredoxins F-acting capping protein and triosephosphate isomerase were previously reported to induce humoral arm of the immune response in cancer
Translationally controlled tumor protein and electron transfer flavoprotein alpha-subunit have not been reported to induce humoral arm of the immune response in cancer so far
In terms of biological processes the identified proteins
belong to several groups
They are mainely involved in cell motility cytoskleton
stress response apoptosis cell signaling and
metabolism
Identification of other immunogenic proteins is
underway
Appel RD et al 1993 - SWISS-2DPAGE a database of two-dimensional gel electrophoresis images Electrophoresis 14 1232-1238
Appel RD Bairoch A Sanchez JC Vargas JR Golaz O Pasquali C and Hochstrasser DF 1996 ndash Federated two-dimensional electrophoresis database a simple means of publishing two-dimensional electrophoresis data Electrophoresis 17 540-546
Bjellqvist B Ek K Righetti PG Gianazza E Gorg A Westermeier R Postel W 1982 ndash Isoelectric focusing in immobilised pH gradients principle methodology and applications JBiochemBiophysMethods 6 317-339
Brazma A et al 2001 ndash Minimum information about a microarray experiment (MIAME)-towards standards for microarray data Nat genetics 29 365-71
Hoogland C Baujard Sanchez JC Hochstrasser DF and Appel RD 1997 ndash Make2ddb a simple package to set up a two-diensional electrophoresis database for the world wide web Electrophoresis 18 2755-2758
OFarrell 1975 - High resolution two-dimensional electrophoresis of proteins JBiolChem 25 250 4007-21
Cancer patients produce
autoantibodies to proteins
that are mutated misfolded
improperly post-translationally
modified overexpressed
truncated or aberrantly localized
in tumor cells
A large number of tumor antigens that elicit autoantibodies have been identified by different methods
Breast cancer is a major cancer cause and death worldwide
Several breast cancer biomarkers such as BRCA estrogen
receptor and HER2 have been shown to have a diagnostic
prognostic or therapeutic value
However they are reliable just in a proportion of patients and
search for finding novel breast cancer biomarkers is underway
The aim of our study was to identify antigens eliciting a humoral
immune response in HER2+ and HER2 NP breast cancers by
two dimensional electrophoresis (2D) Western blotting and
mass spectrometry
Serum samples Our study groups comprised of 18 breast cancer patients
and 9 normal individuals Breast cancer patients had at least one involved
auxiliary lymph node 9 were HER2 negative and 9 were HER2 positive (3+)
Normal individuals had no history of malignancy
autoimmune disease or severe inflammatory disease
The MCF7 (an ER+ low-expressing HER2 cell line) and SKBR3(an ER- high expressing
HER2 cell line) breast cancer cell lines cultured in culture media contained RPMI 1640
medium 10 fetal bovine serum 100UmL penicillin and 100 mgmL streptomycin
Breast cancer cell line was lysed in a lysis solution (7 M Urea 2 M Thoiurea CHAPS 4 DTT
50 mM IPG buffer 1)
Cell lysates were subjected to 2D-PAGE
2D gels were either stained with blue silver method (Staining solution Coomassie Brilliant
blue MeOH orthophosphoric acid ammonium sulfate) or transferred to PVDF membranes
Immuno-reactive protein spots were punched from the gel
and transferred to a 96 well polypropylene plate
Spots were sent for matrix-assisted laser desorptionionisation time-
of-flighttime-of-flight(MALDI-TOFTOF)mass spectrometry
analysis
2 Dimensional Electrophoresis
Proteomics is the large-scale study of proteins
particularly their structures and functions
The proteome is the entire complement of proteins produced by an organism or system This will vary with time
Proteomics is often considered the next step in the study of biological systems after genomics
Why study Proteomics
First the level of transcription of a gene gives only a rough estimate of its level of
expression into a protein An mRNA produced in abundance may be degraded rapidly
or translated inefficiently resulting in a small amount of protein
Second many proteins experience post-translational modifications that profoundly
affect their activities for example some proteins are not active until they become
phosphorylated Methods such as phosphoproteomics and glycoproteomics are used to
study post-translational modifications
Third many transcripts give rise to more than one protein through alternative splicing
or alternative post-translational modifications
Finally At present there is no other technique that is capable of simultaneously
resolving thousands of proteins in one separation procedure
The core technology of proteomics is 2-DE
To identify as many components of the proteome as possible
Protein Mining catalog all the proteins present in a tissue cell organelle etc
Protein Function
Mapping Protein Post-Translational Modification - Characterization of posttranslational modifications phosphorylation glycosylation oxidation etc
Protein Localization
Protein Expression Studies
Comparative proteomics
- proteomics monitors the expression of a large number of proteins within a cell or tissue and observers quantitatively how the
patte of expression changes under different circumstances (comparative proteomics) such as
bull Healthy Diseased
bull Cancerous Benign
bull Drug resistant Drug susceptible
bull Tissue specific
An interesting use of proteomics is using specific protein biomarkers to diagnose disease
Sample Preparation 2-D Electrophoresis Spot Detection amp Image Analysis
Enzymatic Digestion Peptide-Mass Fingerprinting
Peptide Sequencing via MS Database Search
Protein Identification
(I) (III)
(IV) (V) (VI)
(II)
Sample preparation buffer composition IPG strip rehydration 1st dimension IEF run IPG strip wash Equilibration buffer Equilibration buffer 2nd dimension SDS-PAGE Gel staining Immunoblot Imaging
Sample preparation
buffer composition
IPG strip rehydration
1st dimension IEF run
IPG strip wash
Equilibration buffer I
Equilibration buffer II
2nd dimension SDS-PAGE
Gel staining
Imaging
2-D Electrophoresis
Consistent protocol Limit sample degradation
bull fresh cellstissue bull protease inhibitors bull keep sample cold bull long term storage at ndash80degC
Limit sample contamination
bull gloves (keratin)
SolubilisationDenaturation buffer
bull separate proteins into individual components
bull reliable running in the IEF
Gentle lysis method 1 Osmotic lysis (cultured cells)
Suspend cells in hypoosmotic solution
2 Repeated freezing and thawing (bacteria)
Freeze using liquid nitrogen
3 Detergent lysis (yeast and fungi)
Lysis buffer (containing urea and detergent)
SDS (have to be removed before IEF)
4 Enzymatic lysis (plant bacteria fungi)
Lysomzyme (bacteria)
Cellulose and pectinase (plant)
Lyticase (yeast)
Vigorous lysis method
1 Sonication probe (cell suspension)
Avoid overheat cool on ice between burst
2 French pressure (microorganism with cell wall)
Cells are lysed by shear force
3 Mortar and pestle (solid tissue microorganism)
Grind solid tissue to fine powder with liquid nitrogen
4 Sample grinding kit (for small amount of sample)
For precious sample
5 Glass bead (cell suspension microorganism)
Using abrasive vortexed bead to break cell walls
Chaotrope
Urea (up to 9 M)
Thiourea (up to 2 M)
bull Disrupt of hydrogen and hydrophobic bonds
Note Urea (if tdeggt37degC) cyanate (HN=C=O)
carbamylation (Lys Arg)
(R-NH2 R-NH-CO-NH2)
carbamylation trains
Reductants
b-mercaptoethanol
DTT(dithiothreitol)
bull Break disulfide bridge
(within or between protein)
Detergents ndash surfactants
CHAPS (up to 4)
bull Disrupt membranes
bull Break hydrophobic interactions
bull Solubilise lipids
bull Release membrane bound proteins
Note No net charge for IEF (no SDS)
Soluble in urea
Isoelectric Focusing
Measure the protein concenteration in you samples
1 Biuret
2 Lowry methods
3 Bradford methods
4 UV methods
5 Special methods
6 Other commercial methods
BCA assay (bicinchoninic acid assay Pierce)
DC protein assay (detergent compatible Bio-rad)
DCRC protein assay (detergentreducing agent compatible
Bio-rad)
Before runninng IEF you shouldhellip
Introduced by Gorg A
Ref Gorg A (1994) Westermeier (2001)
Dried gel strips can be stored at -20 to -
80 from months to years
Amersham Biosciences Bio-Rad
IPG strips (3 mm x 18 cm x 05 mm)
Narrow range
Medium range
Broad range
4 7
35 45 55 67
40 50 60
3 10
6 11
Load 400 ml sample per groove (no bubbles)
Place IPG strip gel facing down in groove
Add ~25 ml non conductive oil per groove
Peel off protective film from strip
Rehydrate overnight (~22 hrs) at room temperature
Program power supply
bull Number of gels (1-10)
bull Max voltage 5000 V
bull Vhold 125 V
bull Duration 24 hrs 00 min
bull Max current 80 mAstrip
bull Volt hours 80000 Vh
pI
+
+ - - - - - -
+ + + + + + + -
pH 4 pH 7
Cathode Anode
Place wet wicks (DH2O) under each end of strip
Set chiller temperature for 20degC (setting ~25)
Remove IPG strip from tray
Let oil drip off the strip
Place IPG strip gel facing up in equilibration tray
+ -
Add 10 ml equilibration buffer 1 per tray
+ -
+ -
+ -
+ -
(in chemical hood)
MW 4 7
+ -
10 DuracrylTM gel (22 cm x 23 cm x 1 mm)
bull TrisTricineSDS buffer
bull IPG strip
bull MW (gel worm)
bull Agarose overlay
bull Silicone spacer
bull Gasket
-
+
Conditions
bull Number of gel (1-10)
bull Max voltage 500 V
bull Max power 1600 mWgel
bull 4degC (setting 10)
bull ~19 hrs
10 DuracrylTM gel (22 cm x 23 cm x 1 mm)
MW 4 7
+ -
Trisacetate 25 mM Tris-base pH 83 (acetic acid)
200 mM Tris-base 200 mM Tricine 04 SDS
045 mm filtered
TrisTricine SDS
Bio-Rad
Digest to peptide fragment MS analysis
1 First dimension
isoelectric focusing
separation according to the pI
2 Second dimension
SDS electrophoresis (SDS-PAGE)
Separation according to the MW
Interested spot
Sensitivity Process
TimeSteps
Advantages
bullBio-safe Coomassie 10ng 25hr3 steps MS compatible easily visualised non
hazardous
bullCoomassie Blue R-250 40ng 25hr2 steps Oldest and least expensive method
bullSilver Stain Plus 1ng 15hr3 steps MS compatible high sensitivity low
background
bullBio-Rad silver stain 1ng 2hr7 steps High sensitivity detects some highly
glycosylated and other difficult to
stain proteins
bullSypro ruby 1ng 3hr2 steps MS compatible allows analysis in
flourescent imagers linear over 3
orders of magnitude
Coomassie staining
bull moderate sensitivity (36-47 ng)
bull non specific
bull not quantitative
Silver staining
bull sensitive (05-12 ng)
bull time consuming
bull non specific
bull negative stain some spots
Fluorescent dye (SYPRO ruby)
bull sensitive (1-2 ng)
bull specific quantitative
bull end point stain
Coomassie Silver
Coomassie Silver Stain Copper Stain
SDS-polyacrylamide gel
Electrotransfer to membrane
Phosphoimager for 32P and 35S labelled
1D or 2D gels
Fluoroimager for
SYPRO labelled
1D or 2D gels
Densitometer or
Photo Scanner
Normal liver
Tumor
Both
Melanie 4 (GeneBio - Windows only)
httpcaexpasyorgmelanie
ImageMaster 2D Elite (Amersham)
httpwwwimsupportcom
Phoretix 2D Advanced
httpwwwphoretixcom
PDQuest 61 (BioRad - Windows only) httpwwwproteomeworksbio-radcomhtmlpdquesthtml
Mass Spectrometry
wwwexpasych - Swiss-2DPAGE httpwwwanlgovBIOPMG - Mouse liver human breast cell lines pyrococcus Argonne Protein Mapping Group httpwwwharefieldnthamesnhsuknhliproteinindexhtml - HSC-2DPAGE Heart Science Centre Harefield Hospital httpotowustleduthcperi-gelshtm - Washington Univ Inner Ear Protein Database
httpcaexpasyorgch2d2d-indexhtml - World 2DPAGE Index of 2D gel databases
Time line ~ 4 days
bull Protein gt 250 kDa do not enter 2 SDS-PAGE properly
bull PH more than 9
bull PH less than 4
bull Hydrophobic proteins
bull Low abundant proteins
Immunoblotting Proteins from 2D gels were transferred to PVDFmembranes The membranes were blocked with 5 skimmed milk in PBS-Tween (PBS-T) for 3 h The membranes were incubated overnight at 4degC with individual sera diluted 1100 in PBS-T followed by secondary antibody (anti-human IgG conjugated with horseradish peroxidase) for 2 h with dilutions of 13000 Before and after addition of the secondary antibody membranes washed 4 times each time for 5 min in PBS-T To visualize antigenic components membranes were incubated in diaminobenzeden (DAB) and H2O2 for 15 min
List of protein spots that reacted with more than 30 of serum samples
and were identified by mass spectrometry
1Heat shock protein HSP 90-beta
2 precursor(GRP 78) 78 kDa glucose-regulated protein
3 Heat shock cognate 71 kDa protein
4 Stress-70 protein mitochondrial precursor
5 Tubulin beta-5 chain
6Tubulin alpha-6 chain and 60 kDa heat shock protein mitochondrial
precursor (Hsp60)
7 Heterogeneous nuclear ribonucleoprotein K (hnRNP K)
8 Cytokeratin 8
9 Cytokeratin 18
10 Beta-actin
11 cytokeratin 19
12 Eukaryotic initiation factor 4A-I
13 14-3-3 protein epsilon
14 14-3-3 protein zetadelta (Protein kinase Cinhibitor protein-1)
15 Translationally controlled tumor protein (TCTP)
Among these autoantigens cytokeratin 8 cytokeratin 18 Fructose-bisphosphate aldolase and enolase were reacted with more than 60 of healthy individualsrsquo serum samples These antigens were intense spots in gels
Heat Shock proteins tubulins heterogeneous nuclear ribonucleoprotein actins cytokeratins 14-3-3 protein epsilon 14-3-3 protein zetadelta eukaryotic initiation factors Rho GDP-dissociation inhibitors phosphoglycerate mutase 1 G6PD GA3PD peroxiredoxins F-acting capping protein and triosephosphate isomerase were previously reported to induce humoral arm of the immune response in cancer
Translationally controlled tumor protein and electron transfer flavoprotein alpha-subunit have not been reported to induce humoral arm of the immune response in cancer so far
In terms of biological processes the identified proteins
belong to several groups
They are mainely involved in cell motility cytoskleton
stress response apoptosis cell signaling and
metabolism
Identification of other immunogenic proteins is
underway
Appel RD et al 1993 - SWISS-2DPAGE a database of two-dimensional gel electrophoresis images Electrophoresis 14 1232-1238
Appel RD Bairoch A Sanchez JC Vargas JR Golaz O Pasquali C and Hochstrasser DF 1996 ndash Federated two-dimensional electrophoresis database a simple means of publishing two-dimensional electrophoresis data Electrophoresis 17 540-546
Bjellqvist B Ek K Righetti PG Gianazza E Gorg A Westermeier R Postel W 1982 ndash Isoelectric focusing in immobilised pH gradients principle methodology and applications JBiochemBiophysMethods 6 317-339
Brazma A et al 2001 ndash Minimum information about a microarray experiment (MIAME)-towards standards for microarray data Nat genetics 29 365-71
Hoogland C Baujard Sanchez JC Hochstrasser DF and Appel RD 1997 ndash Make2ddb a simple package to set up a two-diensional electrophoresis database for the world wide web Electrophoresis 18 2755-2758
OFarrell 1975 - High resolution two-dimensional electrophoresis of proteins JBiolChem 25 250 4007-21
Breast cancer is a major cancer cause and death worldwide
Several breast cancer biomarkers such as BRCA estrogen
receptor and HER2 have been shown to have a diagnostic
prognostic or therapeutic value
However they are reliable just in a proportion of patients and
search for finding novel breast cancer biomarkers is underway
The aim of our study was to identify antigens eliciting a humoral
immune response in HER2+ and HER2 NP breast cancers by
two dimensional electrophoresis (2D) Western blotting and
mass spectrometry
Serum samples Our study groups comprised of 18 breast cancer patients
and 9 normal individuals Breast cancer patients had at least one involved
auxiliary lymph node 9 were HER2 negative and 9 were HER2 positive (3+)
Normal individuals had no history of malignancy
autoimmune disease or severe inflammatory disease
The MCF7 (an ER+ low-expressing HER2 cell line) and SKBR3(an ER- high expressing
HER2 cell line) breast cancer cell lines cultured in culture media contained RPMI 1640
medium 10 fetal bovine serum 100UmL penicillin and 100 mgmL streptomycin
Breast cancer cell line was lysed in a lysis solution (7 M Urea 2 M Thoiurea CHAPS 4 DTT
50 mM IPG buffer 1)
Cell lysates were subjected to 2D-PAGE
2D gels were either stained with blue silver method (Staining solution Coomassie Brilliant
blue MeOH orthophosphoric acid ammonium sulfate) or transferred to PVDF membranes
Immuno-reactive protein spots were punched from the gel
and transferred to a 96 well polypropylene plate
Spots were sent for matrix-assisted laser desorptionionisation time-
of-flighttime-of-flight(MALDI-TOFTOF)mass spectrometry
analysis
2 Dimensional Electrophoresis
Proteomics is the large-scale study of proteins
particularly their structures and functions
The proteome is the entire complement of proteins produced by an organism or system This will vary with time
Proteomics is often considered the next step in the study of biological systems after genomics
Why study Proteomics
First the level of transcription of a gene gives only a rough estimate of its level of
expression into a protein An mRNA produced in abundance may be degraded rapidly
or translated inefficiently resulting in a small amount of protein
Second many proteins experience post-translational modifications that profoundly
affect their activities for example some proteins are not active until they become
phosphorylated Methods such as phosphoproteomics and glycoproteomics are used to
study post-translational modifications
Third many transcripts give rise to more than one protein through alternative splicing
or alternative post-translational modifications
Finally At present there is no other technique that is capable of simultaneously
resolving thousands of proteins in one separation procedure
The core technology of proteomics is 2-DE
To identify as many components of the proteome as possible
Protein Mining catalog all the proteins present in a tissue cell organelle etc
Protein Function
Mapping Protein Post-Translational Modification - Characterization of posttranslational modifications phosphorylation glycosylation oxidation etc
Protein Localization
Protein Expression Studies
Comparative proteomics
- proteomics monitors the expression of a large number of proteins within a cell or tissue and observers quantitatively how the
patte of expression changes under different circumstances (comparative proteomics) such as
bull Healthy Diseased
bull Cancerous Benign
bull Drug resistant Drug susceptible
bull Tissue specific
An interesting use of proteomics is using specific protein biomarkers to diagnose disease
Sample Preparation 2-D Electrophoresis Spot Detection amp Image Analysis
Enzymatic Digestion Peptide-Mass Fingerprinting
Peptide Sequencing via MS Database Search
Protein Identification
(I) (III)
(IV) (V) (VI)
(II)
Sample preparation buffer composition IPG strip rehydration 1st dimension IEF run IPG strip wash Equilibration buffer Equilibration buffer 2nd dimension SDS-PAGE Gel staining Immunoblot Imaging
Sample preparation
buffer composition
IPG strip rehydration
1st dimension IEF run
IPG strip wash
Equilibration buffer I
Equilibration buffer II
2nd dimension SDS-PAGE
Gel staining
Imaging
2-D Electrophoresis
Consistent protocol Limit sample degradation
bull fresh cellstissue bull protease inhibitors bull keep sample cold bull long term storage at ndash80degC
Limit sample contamination
bull gloves (keratin)
SolubilisationDenaturation buffer
bull separate proteins into individual components
bull reliable running in the IEF
Gentle lysis method 1 Osmotic lysis (cultured cells)
Suspend cells in hypoosmotic solution
2 Repeated freezing and thawing (bacteria)
Freeze using liquid nitrogen
3 Detergent lysis (yeast and fungi)
Lysis buffer (containing urea and detergent)
SDS (have to be removed before IEF)
4 Enzymatic lysis (plant bacteria fungi)
Lysomzyme (bacteria)
Cellulose and pectinase (plant)
Lyticase (yeast)
Vigorous lysis method
1 Sonication probe (cell suspension)
Avoid overheat cool on ice between burst
2 French pressure (microorganism with cell wall)
Cells are lysed by shear force
3 Mortar and pestle (solid tissue microorganism)
Grind solid tissue to fine powder with liquid nitrogen
4 Sample grinding kit (for small amount of sample)
For precious sample
5 Glass bead (cell suspension microorganism)
Using abrasive vortexed bead to break cell walls
Chaotrope
Urea (up to 9 M)
Thiourea (up to 2 M)
bull Disrupt of hydrogen and hydrophobic bonds
Note Urea (if tdeggt37degC) cyanate (HN=C=O)
carbamylation (Lys Arg)
(R-NH2 R-NH-CO-NH2)
carbamylation trains
Reductants
b-mercaptoethanol
DTT(dithiothreitol)
bull Break disulfide bridge
(within or between protein)
Detergents ndash surfactants
CHAPS (up to 4)
bull Disrupt membranes
bull Break hydrophobic interactions
bull Solubilise lipids
bull Release membrane bound proteins
Note No net charge for IEF (no SDS)
Soluble in urea
Isoelectric Focusing
Measure the protein concenteration in you samples
1 Biuret
2 Lowry methods
3 Bradford methods
4 UV methods
5 Special methods
6 Other commercial methods
BCA assay (bicinchoninic acid assay Pierce)
DC protein assay (detergent compatible Bio-rad)
DCRC protein assay (detergentreducing agent compatible
Bio-rad)
Before runninng IEF you shouldhellip
Introduced by Gorg A
Ref Gorg A (1994) Westermeier (2001)
Dried gel strips can be stored at -20 to -
80 from months to years
Amersham Biosciences Bio-Rad
IPG strips (3 mm x 18 cm x 05 mm)
Narrow range
Medium range
Broad range
4 7
35 45 55 67
40 50 60
3 10
6 11
Load 400 ml sample per groove (no bubbles)
Place IPG strip gel facing down in groove
Add ~25 ml non conductive oil per groove
Peel off protective film from strip
Rehydrate overnight (~22 hrs) at room temperature
Program power supply
bull Number of gels (1-10)
bull Max voltage 5000 V
bull Vhold 125 V
bull Duration 24 hrs 00 min
bull Max current 80 mAstrip
bull Volt hours 80000 Vh
pI
+
+ - - - - - -
+ + + + + + + -
pH 4 pH 7
Cathode Anode
Place wet wicks (DH2O) under each end of strip
Set chiller temperature for 20degC (setting ~25)
Remove IPG strip from tray
Let oil drip off the strip
Place IPG strip gel facing up in equilibration tray
+ -
Add 10 ml equilibration buffer 1 per tray
+ -
+ -
+ -
+ -
(in chemical hood)
MW 4 7
+ -
10 DuracrylTM gel (22 cm x 23 cm x 1 mm)
bull TrisTricineSDS buffer
bull IPG strip
bull MW (gel worm)
bull Agarose overlay
bull Silicone spacer
bull Gasket
-
+
Conditions
bull Number of gel (1-10)
bull Max voltage 500 V
bull Max power 1600 mWgel
bull 4degC (setting 10)
bull ~19 hrs
10 DuracrylTM gel (22 cm x 23 cm x 1 mm)
MW 4 7
+ -
Trisacetate 25 mM Tris-base pH 83 (acetic acid)
200 mM Tris-base 200 mM Tricine 04 SDS
045 mm filtered
TrisTricine SDS
Bio-Rad
Digest to peptide fragment MS analysis
1 First dimension
isoelectric focusing
separation according to the pI
2 Second dimension
SDS electrophoresis (SDS-PAGE)
Separation according to the MW
Interested spot
Sensitivity Process
TimeSteps
Advantages
bullBio-safe Coomassie 10ng 25hr3 steps MS compatible easily visualised non
hazardous
bullCoomassie Blue R-250 40ng 25hr2 steps Oldest and least expensive method
bullSilver Stain Plus 1ng 15hr3 steps MS compatible high sensitivity low
background
bullBio-Rad silver stain 1ng 2hr7 steps High sensitivity detects some highly
glycosylated and other difficult to
stain proteins
bullSypro ruby 1ng 3hr2 steps MS compatible allows analysis in
flourescent imagers linear over 3
orders of magnitude
Coomassie staining
bull moderate sensitivity (36-47 ng)
bull non specific
bull not quantitative
Silver staining
bull sensitive (05-12 ng)
bull time consuming
bull non specific
bull negative stain some spots
Fluorescent dye (SYPRO ruby)
bull sensitive (1-2 ng)
bull specific quantitative
bull end point stain
Coomassie Silver
Coomassie Silver Stain Copper Stain
SDS-polyacrylamide gel
Electrotransfer to membrane
Phosphoimager for 32P and 35S labelled
1D or 2D gels
Fluoroimager for
SYPRO labelled
1D or 2D gels
Densitometer or
Photo Scanner
Normal liver
Tumor
Both
Melanie 4 (GeneBio - Windows only)
httpcaexpasyorgmelanie
ImageMaster 2D Elite (Amersham)
httpwwwimsupportcom
Phoretix 2D Advanced
httpwwwphoretixcom
PDQuest 61 (BioRad - Windows only) httpwwwproteomeworksbio-radcomhtmlpdquesthtml
Mass Spectrometry
wwwexpasych - Swiss-2DPAGE httpwwwanlgovBIOPMG - Mouse liver human breast cell lines pyrococcus Argonne Protein Mapping Group httpwwwharefieldnthamesnhsuknhliproteinindexhtml - HSC-2DPAGE Heart Science Centre Harefield Hospital httpotowustleduthcperi-gelshtm - Washington Univ Inner Ear Protein Database
httpcaexpasyorgch2d2d-indexhtml - World 2DPAGE Index of 2D gel databases
Time line ~ 4 days
bull Protein gt 250 kDa do not enter 2 SDS-PAGE properly
bull PH more than 9
bull PH less than 4
bull Hydrophobic proteins
bull Low abundant proteins
Immunoblotting Proteins from 2D gels were transferred to PVDFmembranes The membranes were blocked with 5 skimmed milk in PBS-Tween (PBS-T) for 3 h The membranes were incubated overnight at 4degC with individual sera diluted 1100 in PBS-T followed by secondary antibody (anti-human IgG conjugated with horseradish peroxidase) for 2 h with dilutions of 13000 Before and after addition of the secondary antibody membranes washed 4 times each time for 5 min in PBS-T To visualize antigenic components membranes were incubated in diaminobenzeden (DAB) and H2O2 for 15 min
List of protein spots that reacted with more than 30 of serum samples
and were identified by mass spectrometry
1Heat shock protein HSP 90-beta
2 precursor(GRP 78) 78 kDa glucose-regulated protein
3 Heat shock cognate 71 kDa protein
4 Stress-70 protein mitochondrial precursor
5 Tubulin beta-5 chain
6Tubulin alpha-6 chain and 60 kDa heat shock protein mitochondrial
precursor (Hsp60)
7 Heterogeneous nuclear ribonucleoprotein K (hnRNP K)
8 Cytokeratin 8
9 Cytokeratin 18
10 Beta-actin
11 cytokeratin 19
12 Eukaryotic initiation factor 4A-I
13 14-3-3 protein epsilon
14 14-3-3 protein zetadelta (Protein kinase Cinhibitor protein-1)
15 Translationally controlled tumor protein (TCTP)
Among these autoantigens cytokeratin 8 cytokeratin 18 Fructose-bisphosphate aldolase and enolase were reacted with more than 60 of healthy individualsrsquo serum samples These antigens were intense spots in gels
Heat Shock proteins tubulins heterogeneous nuclear ribonucleoprotein actins cytokeratins 14-3-3 protein epsilon 14-3-3 protein zetadelta eukaryotic initiation factors Rho GDP-dissociation inhibitors phosphoglycerate mutase 1 G6PD GA3PD peroxiredoxins F-acting capping protein and triosephosphate isomerase were previously reported to induce humoral arm of the immune response in cancer
Translationally controlled tumor protein and electron transfer flavoprotein alpha-subunit have not been reported to induce humoral arm of the immune response in cancer so far
In terms of biological processes the identified proteins
belong to several groups
They are mainely involved in cell motility cytoskleton
stress response apoptosis cell signaling and
metabolism
Identification of other immunogenic proteins is
underway
Appel RD et al 1993 - SWISS-2DPAGE a database of two-dimensional gel electrophoresis images Electrophoresis 14 1232-1238
Appel RD Bairoch A Sanchez JC Vargas JR Golaz O Pasquali C and Hochstrasser DF 1996 ndash Federated two-dimensional electrophoresis database a simple means of publishing two-dimensional electrophoresis data Electrophoresis 17 540-546
Bjellqvist B Ek K Righetti PG Gianazza E Gorg A Westermeier R Postel W 1982 ndash Isoelectric focusing in immobilised pH gradients principle methodology and applications JBiochemBiophysMethods 6 317-339
Brazma A et al 2001 ndash Minimum information about a microarray experiment (MIAME)-towards standards for microarray data Nat genetics 29 365-71
Hoogland C Baujard Sanchez JC Hochstrasser DF and Appel RD 1997 ndash Make2ddb a simple package to set up a two-diensional electrophoresis database for the world wide web Electrophoresis 18 2755-2758
OFarrell 1975 - High resolution two-dimensional electrophoresis of proteins JBiolChem 25 250 4007-21
Serum samples Our study groups comprised of 18 breast cancer patients
and 9 normal individuals Breast cancer patients had at least one involved
auxiliary lymph node 9 were HER2 negative and 9 were HER2 positive (3+)
Normal individuals had no history of malignancy
autoimmune disease or severe inflammatory disease
The MCF7 (an ER+ low-expressing HER2 cell line) and SKBR3(an ER- high expressing
HER2 cell line) breast cancer cell lines cultured in culture media contained RPMI 1640
medium 10 fetal bovine serum 100UmL penicillin and 100 mgmL streptomycin
Breast cancer cell line was lysed in a lysis solution (7 M Urea 2 M Thoiurea CHAPS 4 DTT
50 mM IPG buffer 1)
Cell lysates were subjected to 2D-PAGE
2D gels were either stained with blue silver method (Staining solution Coomassie Brilliant
blue MeOH orthophosphoric acid ammonium sulfate) or transferred to PVDF membranes
Immuno-reactive protein spots were punched from the gel
and transferred to a 96 well polypropylene plate
Spots were sent for matrix-assisted laser desorptionionisation time-
of-flighttime-of-flight(MALDI-TOFTOF)mass spectrometry
analysis
2 Dimensional Electrophoresis
Proteomics is the large-scale study of proteins
particularly their structures and functions
The proteome is the entire complement of proteins produced by an organism or system This will vary with time
Proteomics is often considered the next step in the study of biological systems after genomics
Why study Proteomics
First the level of transcription of a gene gives only a rough estimate of its level of
expression into a protein An mRNA produced in abundance may be degraded rapidly
or translated inefficiently resulting in a small amount of protein
Second many proteins experience post-translational modifications that profoundly
affect their activities for example some proteins are not active until they become
phosphorylated Methods such as phosphoproteomics and glycoproteomics are used to
study post-translational modifications
Third many transcripts give rise to more than one protein through alternative splicing
or alternative post-translational modifications
Finally At present there is no other technique that is capable of simultaneously
resolving thousands of proteins in one separation procedure
The core technology of proteomics is 2-DE
To identify as many components of the proteome as possible
Protein Mining catalog all the proteins present in a tissue cell organelle etc
Protein Function
Mapping Protein Post-Translational Modification - Characterization of posttranslational modifications phosphorylation glycosylation oxidation etc
Protein Localization
Protein Expression Studies
Comparative proteomics
- proteomics monitors the expression of a large number of proteins within a cell or tissue and observers quantitatively how the
patte of expression changes under different circumstances (comparative proteomics) such as
bull Healthy Diseased
bull Cancerous Benign
bull Drug resistant Drug susceptible
bull Tissue specific
An interesting use of proteomics is using specific protein biomarkers to diagnose disease
Sample Preparation 2-D Electrophoresis Spot Detection amp Image Analysis
Enzymatic Digestion Peptide-Mass Fingerprinting
Peptide Sequencing via MS Database Search
Protein Identification
(I) (III)
(IV) (V) (VI)
(II)
Sample preparation buffer composition IPG strip rehydration 1st dimension IEF run IPG strip wash Equilibration buffer Equilibration buffer 2nd dimension SDS-PAGE Gel staining Immunoblot Imaging
Sample preparation
buffer composition
IPG strip rehydration
1st dimension IEF run
IPG strip wash
Equilibration buffer I
Equilibration buffer II
2nd dimension SDS-PAGE
Gel staining
Imaging
2-D Electrophoresis
Consistent protocol Limit sample degradation
bull fresh cellstissue bull protease inhibitors bull keep sample cold bull long term storage at ndash80degC
Limit sample contamination
bull gloves (keratin)
SolubilisationDenaturation buffer
bull separate proteins into individual components
bull reliable running in the IEF
Gentle lysis method 1 Osmotic lysis (cultured cells)
Suspend cells in hypoosmotic solution
2 Repeated freezing and thawing (bacteria)
Freeze using liquid nitrogen
3 Detergent lysis (yeast and fungi)
Lysis buffer (containing urea and detergent)
SDS (have to be removed before IEF)
4 Enzymatic lysis (plant bacteria fungi)
Lysomzyme (bacteria)
Cellulose and pectinase (plant)
Lyticase (yeast)
Vigorous lysis method
1 Sonication probe (cell suspension)
Avoid overheat cool on ice between burst
2 French pressure (microorganism with cell wall)
Cells are lysed by shear force
3 Mortar and pestle (solid tissue microorganism)
Grind solid tissue to fine powder with liquid nitrogen
4 Sample grinding kit (for small amount of sample)
For precious sample
5 Glass bead (cell suspension microorganism)
Using abrasive vortexed bead to break cell walls
Chaotrope
Urea (up to 9 M)
Thiourea (up to 2 M)
bull Disrupt of hydrogen and hydrophobic bonds
Note Urea (if tdeggt37degC) cyanate (HN=C=O)
carbamylation (Lys Arg)
(R-NH2 R-NH-CO-NH2)
carbamylation trains
Reductants
b-mercaptoethanol
DTT(dithiothreitol)
bull Break disulfide bridge
(within or between protein)
Detergents ndash surfactants
CHAPS (up to 4)
bull Disrupt membranes
bull Break hydrophobic interactions
bull Solubilise lipids
bull Release membrane bound proteins
Note No net charge for IEF (no SDS)
Soluble in urea
Isoelectric Focusing
Measure the protein concenteration in you samples
1 Biuret
2 Lowry methods
3 Bradford methods
4 UV methods
5 Special methods
6 Other commercial methods
BCA assay (bicinchoninic acid assay Pierce)
DC protein assay (detergent compatible Bio-rad)
DCRC protein assay (detergentreducing agent compatible
Bio-rad)
Before runninng IEF you shouldhellip
Introduced by Gorg A
Ref Gorg A (1994) Westermeier (2001)
Dried gel strips can be stored at -20 to -
80 from months to years
Amersham Biosciences Bio-Rad
IPG strips (3 mm x 18 cm x 05 mm)
Narrow range
Medium range
Broad range
4 7
35 45 55 67
40 50 60
3 10
6 11
Load 400 ml sample per groove (no bubbles)
Place IPG strip gel facing down in groove
Add ~25 ml non conductive oil per groove
Peel off protective film from strip
Rehydrate overnight (~22 hrs) at room temperature
Program power supply
bull Number of gels (1-10)
bull Max voltage 5000 V
bull Vhold 125 V
bull Duration 24 hrs 00 min
bull Max current 80 mAstrip
bull Volt hours 80000 Vh
pI
+
+ - - - - - -
+ + + + + + + -
pH 4 pH 7
Cathode Anode
Place wet wicks (DH2O) under each end of strip
Set chiller temperature for 20degC (setting ~25)
Remove IPG strip from tray
Let oil drip off the strip
Place IPG strip gel facing up in equilibration tray
+ -
Add 10 ml equilibration buffer 1 per tray
+ -
+ -
+ -
+ -
(in chemical hood)
MW 4 7
+ -
10 DuracrylTM gel (22 cm x 23 cm x 1 mm)
bull TrisTricineSDS buffer
bull IPG strip
bull MW (gel worm)
bull Agarose overlay
bull Silicone spacer
bull Gasket
-
+
Conditions
bull Number of gel (1-10)
bull Max voltage 500 V
bull Max power 1600 mWgel
bull 4degC (setting 10)
bull ~19 hrs
10 DuracrylTM gel (22 cm x 23 cm x 1 mm)
MW 4 7
+ -
Trisacetate 25 mM Tris-base pH 83 (acetic acid)
200 mM Tris-base 200 mM Tricine 04 SDS
045 mm filtered
TrisTricine SDS
Bio-Rad
Digest to peptide fragment MS analysis
1 First dimension
isoelectric focusing
separation according to the pI
2 Second dimension
SDS electrophoresis (SDS-PAGE)
Separation according to the MW
Interested spot
Sensitivity Process
TimeSteps
Advantages
bullBio-safe Coomassie 10ng 25hr3 steps MS compatible easily visualised non
hazardous
bullCoomassie Blue R-250 40ng 25hr2 steps Oldest and least expensive method
bullSilver Stain Plus 1ng 15hr3 steps MS compatible high sensitivity low
background
bullBio-Rad silver stain 1ng 2hr7 steps High sensitivity detects some highly
glycosylated and other difficult to
stain proteins
bullSypro ruby 1ng 3hr2 steps MS compatible allows analysis in
flourescent imagers linear over 3
orders of magnitude
Coomassie staining
bull moderate sensitivity (36-47 ng)
bull non specific
bull not quantitative
Silver staining
bull sensitive (05-12 ng)
bull time consuming
bull non specific
bull negative stain some spots
Fluorescent dye (SYPRO ruby)
bull sensitive (1-2 ng)
bull specific quantitative
bull end point stain
Coomassie Silver
Coomassie Silver Stain Copper Stain
SDS-polyacrylamide gel
Electrotransfer to membrane
Phosphoimager for 32P and 35S labelled
1D or 2D gels
Fluoroimager for
SYPRO labelled
1D or 2D gels
Densitometer or
Photo Scanner
Normal liver
Tumor
Both
Melanie 4 (GeneBio - Windows only)
httpcaexpasyorgmelanie
ImageMaster 2D Elite (Amersham)
httpwwwimsupportcom
Phoretix 2D Advanced
httpwwwphoretixcom
PDQuest 61 (BioRad - Windows only) httpwwwproteomeworksbio-radcomhtmlpdquesthtml
Mass Spectrometry
wwwexpasych - Swiss-2DPAGE httpwwwanlgovBIOPMG - Mouse liver human breast cell lines pyrococcus Argonne Protein Mapping Group httpwwwharefieldnthamesnhsuknhliproteinindexhtml - HSC-2DPAGE Heart Science Centre Harefield Hospital httpotowustleduthcperi-gelshtm - Washington Univ Inner Ear Protein Database
httpcaexpasyorgch2d2d-indexhtml - World 2DPAGE Index of 2D gel databases
Time line ~ 4 days
bull Protein gt 250 kDa do not enter 2 SDS-PAGE properly
bull PH more than 9
bull PH less than 4
bull Hydrophobic proteins
bull Low abundant proteins
Immunoblotting Proteins from 2D gels were transferred to PVDFmembranes The membranes were blocked with 5 skimmed milk in PBS-Tween (PBS-T) for 3 h The membranes were incubated overnight at 4degC with individual sera diluted 1100 in PBS-T followed by secondary antibody (anti-human IgG conjugated with horseradish peroxidase) for 2 h with dilutions of 13000 Before and after addition of the secondary antibody membranes washed 4 times each time for 5 min in PBS-T To visualize antigenic components membranes were incubated in diaminobenzeden (DAB) and H2O2 for 15 min
List of protein spots that reacted with more than 30 of serum samples
and were identified by mass spectrometry
1Heat shock protein HSP 90-beta
2 precursor(GRP 78) 78 kDa glucose-regulated protein
3 Heat shock cognate 71 kDa protein
4 Stress-70 protein mitochondrial precursor
5 Tubulin beta-5 chain
6Tubulin alpha-6 chain and 60 kDa heat shock protein mitochondrial
precursor (Hsp60)
7 Heterogeneous nuclear ribonucleoprotein K (hnRNP K)
8 Cytokeratin 8
9 Cytokeratin 18
10 Beta-actin
11 cytokeratin 19
12 Eukaryotic initiation factor 4A-I
13 14-3-3 protein epsilon
14 14-3-3 protein zetadelta (Protein kinase Cinhibitor protein-1)
15 Translationally controlled tumor protein (TCTP)
Among these autoantigens cytokeratin 8 cytokeratin 18 Fructose-bisphosphate aldolase and enolase were reacted with more than 60 of healthy individualsrsquo serum samples These antigens were intense spots in gels
Heat Shock proteins tubulins heterogeneous nuclear ribonucleoprotein actins cytokeratins 14-3-3 protein epsilon 14-3-3 protein zetadelta eukaryotic initiation factors Rho GDP-dissociation inhibitors phosphoglycerate mutase 1 G6PD GA3PD peroxiredoxins F-acting capping protein and triosephosphate isomerase were previously reported to induce humoral arm of the immune response in cancer
Translationally controlled tumor protein and electron transfer flavoprotein alpha-subunit have not been reported to induce humoral arm of the immune response in cancer so far
In terms of biological processes the identified proteins
belong to several groups
They are mainely involved in cell motility cytoskleton
stress response apoptosis cell signaling and
metabolism
Identification of other immunogenic proteins is
underway
Appel RD et al 1993 - SWISS-2DPAGE a database of two-dimensional gel electrophoresis images Electrophoresis 14 1232-1238
Appel RD Bairoch A Sanchez JC Vargas JR Golaz O Pasquali C and Hochstrasser DF 1996 ndash Federated two-dimensional electrophoresis database a simple means of publishing two-dimensional electrophoresis data Electrophoresis 17 540-546
Bjellqvist B Ek K Righetti PG Gianazza E Gorg A Westermeier R Postel W 1982 ndash Isoelectric focusing in immobilised pH gradients principle methodology and applications JBiochemBiophysMethods 6 317-339
Brazma A et al 2001 ndash Minimum information about a microarray experiment (MIAME)-towards standards for microarray data Nat genetics 29 365-71
Hoogland C Baujard Sanchez JC Hochstrasser DF and Appel RD 1997 ndash Make2ddb a simple package to set up a two-diensional electrophoresis database for the world wide web Electrophoresis 18 2755-2758
OFarrell 1975 - High resolution two-dimensional electrophoresis of proteins JBiolChem 25 250 4007-21
The MCF7 (an ER+ low-expressing HER2 cell line) and SKBR3(an ER- high expressing
HER2 cell line) breast cancer cell lines cultured in culture media contained RPMI 1640
medium 10 fetal bovine serum 100UmL penicillin and 100 mgmL streptomycin
Breast cancer cell line was lysed in a lysis solution (7 M Urea 2 M Thoiurea CHAPS 4 DTT
50 mM IPG buffer 1)
Cell lysates were subjected to 2D-PAGE
2D gels were either stained with blue silver method (Staining solution Coomassie Brilliant
blue MeOH orthophosphoric acid ammonium sulfate) or transferred to PVDF membranes
Immuno-reactive protein spots were punched from the gel
and transferred to a 96 well polypropylene plate
Spots were sent for matrix-assisted laser desorptionionisation time-
of-flighttime-of-flight(MALDI-TOFTOF)mass spectrometry
analysis
2 Dimensional Electrophoresis
Proteomics is the large-scale study of proteins
particularly their structures and functions
The proteome is the entire complement of proteins produced by an organism or system This will vary with time
Proteomics is often considered the next step in the study of biological systems after genomics
Why study Proteomics
First the level of transcription of a gene gives only a rough estimate of its level of
expression into a protein An mRNA produced in abundance may be degraded rapidly
or translated inefficiently resulting in a small amount of protein
Second many proteins experience post-translational modifications that profoundly
affect their activities for example some proteins are not active until they become
phosphorylated Methods such as phosphoproteomics and glycoproteomics are used to
study post-translational modifications
Third many transcripts give rise to more than one protein through alternative splicing
or alternative post-translational modifications
Finally At present there is no other technique that is capable of simultaneously
resolving thousands of proteins in one separation procedure
The core technology of proteomics is 2-DE
To identify as many components of the proteome as possible
Protein Mining catalog all the proteins present in a tissue cell organelle etc
Protein Function
Mapping Protein Post-Translational Modification - Characterization of posttranslational modifications phosphorylation glycosylation oxidation etc
Protein Localization
Protein Expression Studies
Comparative proteomics
- proteomics monitors the expression of a large number of proteins within a cell or tissue and observers quantitatively how the
patte of expression changes under different circumstances (comparative proteomics) such as
bull Healthy Diseased
bull Cancerous Benign
bull Drug resistant Drug susceptible
bull Tissue specific
An interesting use of proteomics is using specific protein biomarkers to diagnose disease
Sample Preparation 2-D Electrophoresis Spot Detection amp Image Analysis
Enzymatic Digestion Peptide-Mass Fingerprinting
Peptide Sequencing via MS Database Search
Protein Identification
(I) (III)
(IV) (V) (VI)
(II)
Sample preparation buffer composition IPG strip rehydration 1st dimension IEF run IPG strip wash Equilibration buffer Equilibration buffer 2nd dimension SDS-PAGE Gel staining Immunoblot Imaging
Sample preparation
buffer composition
IPG strip rehydration
1st dimension IEF run
IPG strip wash
Equilibration buffer I
Equilibration buffer II
2nd dimension SDS-PAGE
Gel staining
Imaging
2-D Electrophoresis
Consistent protocol Limit sample degradation
bull fresh cellstissue bull protease inhibitors bull keep sample cold bull long term storage at ndash80degC
Limit sample contamination
bull gloves (keratin)
SolubilisationDenaturation buffer
bull separate proteins into individual components
bull reliable running in the IEF
Gentle lysis method 1 Osmotic lysis (cultured cells)
Suspend cells in hypoosmotic solution
2 Repeated freezing and thawing (bacteria)
Freeze using liquid nitrogen
3 Detergent lysis (yeast and fungi)
Lysis buffer (containing urea and detergent)
SDS (have to be removed before IEF)
4 Enzymatic lysis (plant bacteria fungi)
Lysomzyme (bacteria)
Cellulose and pectinase (plant)
Lyticase (yeast)
Vigorous lysis method
1 Sonication probe (cell suspension)
Avoid overheat cool on ice between burst
2 French pressure (microorganism with cell wall)
Cells are lysed by shear force
3 Mortar and pestle (solid tissue microorganism)
Grind solid tissue to fine powder with liquid nitrogen
4 Sample grinding kit (for small amount of sample)
For precious sample
5 Glass bead (cell suspension microorganism)
Using abrasive vortexed bead to break cell walls
Chaotrope
Urea (up to 9 M)
Thiourea (up to 2 M)
bull Disrupt of hydrogen and hydrophobic bonds
Note Urea (if tdeggt37degC) cyanate (HN=C=O)
carbamylation (Lys Arg)
(R-NH2 R-NH-CO-NH2)
carbamylation trains
Reductants
b-mercaptoethanol
DTT(dithiothreitol)
bull Break disulfide bridge
(within or between protein)
Detergents ndash surfactants
CHAPS (up to 4)
bull Disrupt membranes
bull Break hydrophobic interactions
bull Solubilise lipids
bull Release membrane bound proteins
Note No net charge for IEF (no SDS)
Soluble in urea
Isoelectric Focusing
Measure the protein concenteration in you samples
1 Biuret
2 Lowry methods
3 Bradford methods
4 UV methods
5 Special methods
6 Other commercial methods
BCA assay (bicinchoninic acid assay Pierce)
DC protein assay (detergent compatible Bio-rad)
DCRC protein assay (detergentreducing agent compatible
Bio-rad)
Before runninng IEF you shouldhellip
Introduced by Gorg A
Ref Gorg A (1994) Westermeier (2001)
Dried gel strips can be stored at -20 to -
80 from months to years
Amersham Biosciences Bio-Rad
IPG strips (3 mm x 18 cm x 05 mm)
Narrow range
Medium range
Broad range
4 7
35 45 55 67
40 50 60
3 10
6 11
Load 400 ml sample per groove (no bubbles)
Place IPG strip gel facing down in groove
Add ~25 ml non conductive oil per groove
Peel off protective film from strip
Rehydrate overnight (~22 hrs) at room temperature
Program power supply
bull Number of gels (1-10)
bull Max voltage 5000 V
bull Vhold 125 V
bull Duration 24 hrs 00 min
bull Max current 80 mAstrip
bull Volt hours 80000 Vh
pI
+
+ - - - - - -
+ + + + + + + -
pH 4 pH 7
Cathode Anode
Place wet wicks (DH2O) under each end of strip
Set chiller temperature for 20degC (setting ~25)
Remove IPG strip from tray
Let oil drip off the strip
Place IPG strip gel facing up in equilibration tray
+ -
Add 10 ml equilibration buffer 1 per tray
+ -
+ -
+ -
+ -
(in chemical hood)
MW 4 7
+ -
10 DuracrylTM gel (22 cm x 23 cm x 1 mm)
bull TrisTricineSDS buffer
bull IPG strip
bull MW (gel worm)
bull Agarose overlay
bull Silicone spacer
bull Gasket
-
+
Conditions
bull Number of gel (1-10)
bull Max voltage 500 V
bull Max power 1600 mWgel
bull 4degC (setting 10)
bull ~19 hrs
10 DuracrylTM gel (22 cm x 23 cm x 1 mm)
MW 4 7
+ -
Trisacetate 25 mM Tris-base pH 83 (acetic acid)
200 mM Tris-base 200 mM Tricine 04 SDS
045 mm filtered
TrisTricine SDS
Bio-Rad
Digest to peptide fragment MS analysis
1 First dimension
isoelectric focusing
separation according to the pI
2 Second dimension
SDS electrophoresis (SDS-PAGE)
Separation according to the MW
Interested spot
Sensitivity Process
TimeSteps
Advantages
bullBio-safe Coomassie 10ng 25hr3 steps MS compatible easily visualised non
hazardous
bullCoomassie Blue R-250 40ng 25hr2 steps Oldest and least expensive method
bullSilver Stain Plus 1ng 15hr3 steps MS compatible high sensitivity low
background
bullBio-Rad silver stain 1ng 2hr7 steps High sensitivity detects some highly
glycosylated and other difficult to
stain proteins
bullSypro ruby 1ng 3hr2 steps MS compatible allows analysis in
flourescent imagers linear over 3
orders of magnitude
Coomassie staining
bull moderate sensitivity (36-47 ng)
bull non specific
bull not quantitative
Silver staining
bull sensitive (05-12 ng)
bull time consuming
bull non specific
bull negative stain some spots
Fluorescent dye (SYPRO ruby)
bull sensitive (1-2 ng)
bull specific quantitative
bull end point stain
Coomassie Silver
Coomassie Silver Stain Copper Stain
SDS-polyacrylamide gel
Electrotransfer to membrane
Phosphoimager for 32P and 35S labelled
1D or 2D gels
Fluoroimager for
SYPRO labelled
1D or 2D gels
Densitometer or
Photo Scanner
Normal liver
Tumor
Both
Melanie 4 (GeneBio - Windows only)
httpcaexpasyorgmelanie
ImageMaster 2D Elite (Amersham)
httpwwwimsupportcom
Phoretix 2D Advanced
httpwwwphoretixcom
PDQuest 61 (BioRad - Windows only) httpwwwproteomeworksbio-radcomhtmlpdquesthtml
Mass Spectrometry
wwwexpasych - Swiss-2DPAGE httpwwwanlgovBIOPMG - Mouse liver human breast cell lines pyrococcus Argonne Protein Mapping Group httpwwwharefieldnthamesnhsuknhliproteinindexhtml - HSC-2DPAGE Heart Science Centre Harefield Hospital httpotowustleduthcperi-gelshtm - Washington Univ Inner Ear Protein Database
httpcaexpasyorgch2d2d-indexhtml - World 2DPAGE Index of 2D gel databases
Time line ~ 4 days
bull Protein gt 250 kDa do not enter 2 SDS-PAGE properly
bull PH more than 9
bull PH less than 4
bull Hydrophobic proteins
bull Low abundant proteins
Immunoblotting Proteins from 2D gels were transferred to PVDFmembranes The membranes were blocked with 5 skimmed milk in PBS-Tween (PBS-T) for 3 h The membranes were incubated overnight at 4degC with individual sera diluted 1100 in PBS-T followed by secondary antibody (anti-human IgG conjugated with horseradish peroxidase) for 2 h with dilutions of 13000 Before and after addition of the secondary antibody membranes washed 4 times each time for 5 min in PBS-T To visualize antigenic components membranes were incubated in diaminobenzeden (DAB) and H2O2 for 15 min
List of protein spots that reacted with more than 30 of serum samples
and were identified by mass spectrometry
1Heat shock protein HSP 90-beta
2 precursor(GRP 78) 78 kDa glucose-regulated protein
3 Heat shock cognate 71 kDa protein
4 Stress-70 protein mitochondrial precursor
5 Tubulin beta-5 chain
6Tubulin alpha-6 chain and 60 kDa heat shock protein mitochondrial
precursor (Hsp60)
7 Heterogeneous nuclear ribonucleoprotein K (hnRNP K)
8 Cytokeratin 8
9 Cytokeratin 18
10 Beta-actin
11 cytokeratin 19
12 Eukaryotic initiation factor 4A-I
13 14-3-3 protein epsilon
14 14-3-3 protein zetadelta (Protein kinase Cinhibitor protein-1)
15 Translationally controlled tumor protein (TCTP)
Among these autoantigens cytokeratin 8 cytokeratin 18 Fructose-bisphosphate aldolase and enolase were reacted with more than 60 of healthy individualsrsquo serum samples These antigens were intense spots in gels
Heat Shock proteins tubulins heterogeneous nuclear ribonucleoprotein actins cytokeratins 14-3-3 protein epsilon 14-3-3 protein zetadelta eukaryotic initiation factors Rho GDP-dissociation inhibitors phosphoglycerate mutase 1 G6PD GA3PD peroxiredoxins F-acting capping protein and triosephosphate isomerase were previously reported to induce humoral arm of the immune response in cancer
Translationally controlled tumor protein and electron transfer flavoprotein alpha-subunit have not been reported to induce humoral arm of the immune response in cancer so far
In terms of biological processes the identified proteins
belong to several groups
They are mainely involved in cell motility cytoskleton
stress response apoptosis cell signaling and
metabolism
Identification of other immunogenic proteins is
underway
Appel RD et al 1993 - SWISS-2DPAGE a database of two-dimensional gel electrophoresis images Electrophoresis 14 1232-1238
Appel RD Bairoch A Sanchez JC Vargas JR Golaz O Pasquali C and Hochstrasser DF 1996 ndash Federated two-dimensional electrophoresis database a simple means of publishing two-dimensional electrophoresis data Electrophoresis 17 540-546
Bjellqvist B Ek K Righetti PG Gianazza E Gorg A Westermeier R Postel W 1982 ndash Isoelectric focusing in immobilised pH gradients principle methodology and applications JBiochemBiophysMethods 6 317-339
Brazma A et al 2001 ndash Minimum information about a microarray experiment (MIAME)-towards standards for microarray data Nat genetics 29 365-71
Hoogland C Baujard Sanchez JC Hochstrasser DF and Appel RD 1997 ndash Make2ddb a simple package to set up a two-diensional electrophoresis database for the world wide web Electrophoresis 18 2755-2758
OFarrell 1975 - High resolution two-dimensional electrophoresis of proteins JBiolChem 25 250 4007-21
Immuno-reactive protein spots were punched from the gel
and transferred to a 96 well polypropylene plate
Spots were sent for matrix-assisted laser desorptionionisation time-
of-flighttime-of-flight(MALDI-TOFTOF)mass spectrometry
analysis
2 Dimensional Electrophoresis
Proteomics is the large-scale study of proteins
particularly their structures and functions
The proteome is the entire complement of proteins produced by an organism or system This will vary with time
Proteomics is often considered the next step in the study of biological systems after genomics
Why study Proteomics
First the level of transcription of a gene gives only a rough estimate of its level of
expression into a protein An mRNA produced in abundance may be degraded rapidly
or translated inefficiently resulting in a small amount of protein
Second many proteins experience post-translational modifications that profoundly
affect their activities for example some proteins are not active until they become
phosphorylated Methods such as phosphoproteomics and glycoproteomics are used to
study post-translational modifications
Third many transcripts give rise to more than one protein through alternative splicing
or alternative post-translational modifications
Finally At present there is no other technique that is capable of simultaneously
resolving thousands of proteins in one separation procedure
The core technology of proteomics is 2-DE
To identify as many components of the proteome as possible
Protein Mining catalog all the proteins present in a tissue cell organelle etc
Protein Function
Mapping Protein Post-Translational Modification - Characterization of posttranslational modifications phosphorylation glycosylation oxidation etc
Protein Localization
Protein Expression Studies
Comparative proteomics
- proteomics monitors the expression of a large number of proteins within a cell or tissue and observers quantitatively how the
patte of expression changes under different circumstances (comparative proteomics) such as
bull Healthy Diseased
bull Cancerous Benign
bull Drug resistant Drug susceptible
bull Tissue specific
An interesting use of proteomics is using specific protein biomarkers to diagnose disease
Sample Preparation 2-D Electrophoresis Spot Detection amp Image Analysis
Enzymatic Digestion Peptide-Mass Fingerprinting
Peptide Sequencing via MS Database Search
Protein Identification
(I) (III)
(IV) (V) (VI)
(II)
Sample preparation buffer composition IPG strip rehydration 1st dimension IEF run IPG strip wash Equilibration buffer Equilibration buffer 2nd dimension SDS-PAGE Gel staining Immunoblot Imaging
Sample preparation
buffer composition
IPG strip rehydration
1st dimension IEF run
IPG strip wash
Equilibration buffer I
Equilibration buffer II
2nd dimension SDS-PAGE
Gel staining
Imaging
2-D Electrophoresis
Consistent protocol Limit sample degradation
bull fresh cellstissue bull protease inhibitors bull keep sample cold bull long term storage at ndash80degC
Limit sample contamination
bull gloves (keratin)
SolubilisationDenaturation buffer
bull separate proteins into individual components
bull reliable running in the IEF
Gentle lysis method 1 Osmotic lysis (cultured cells)
Suspend cells in hypoosmotic solution
2 Repeated freezing and thawing (bacteria)
Freeze using liquid nitrogen
3 Detergent lysis (yeast and fungi)
Lysis buffer (containing urea and detergent)
SDS (have to be removed before IEF)
4 Enzymatic lysis (plant bacteria fungi)
Lysomzyme (bacteria)
Cellulose and pectinase (plant)
Lyticase (yeast)
Vigorous lysis method
1 Sonication probe (cell suspension)
Avoid overheat cool on ice between burst
2 French pressure (microorganism with cell wall)
Cells are lysed by shear force
3 Mortar and pestle (solid tissue microorganism)
Grind solid tissue to fine powder with liquid nitrogen
4 Sample grinding kit (for small amount of sample)
For precious sample
5 Glass bead (cell suspension microorganism)
Using abrasive vortexed bead to break cell walls
Chaotrope
Urea (up to 9 M)
Thiourea (up to 2 M)
bull Disrupt of hydrogen and hydrophobic bonds
Note Urea (if tdeggt37degC) cyanate (HN=C=O)
carbamylation (Lys Arg)
(R-NH2 R-NH-CO-NH2)
carbamylation trains
Reductants
b-mercaptoethanol
DTT(dithiothreitol)
bull Break disulfide bridge
(within or between protein)
Detergents ndash surfactants
CHAPS (up to 4)
bull Disrupt membranes
bull Break hydrophobic interactions
bull Solubilise lipids
bull Release membrane bound proteins
Note No net charge for IEF (no SDS)
Soluble in urea
Isoelectric Focusing
Measure the protein concenteration in you samples
1 Biuret
2 Lowry methods
3 Bradford methods
4 UV methods
5 Special methods
6 Other commercial methods
BCA assay (bicinchoninic acid assay Pierce)
DC protein assay (detergent compatible Bio-rad)
DCRC protein assay (detergentreducing agent compatible
Bio-rad)
Before runninng IEF you shouldhellip
Introduced by Gorg A
Ref Gorg A (1994) Westermeier (2001)
Dried gel strips can be stored at -20 to -
80 from months to years
Amersham Biosciences Bio-Rad
IPG strips (3 mm x 18 cm x 05 mm)
Narrow range
Medium range
Broad range
4 7
35 45 55 67
40 50 60
3 10
6 11
Load 400 ml sample per groove (no bubbles)
Place IPG strip gel facing down in groove
Add ~25 ml non conductive oil per groove
Peel off protective film from strip
Rehydrate overnight (~22 hrs) at room temperature
Program power supply
bull Number of gels (1-10)
bull Max voltage 5000 V
bull Vhold 125 V
bull Duration 24 hrs 00 min
bull Max current 80 mAstrip
bull Volt hours 80000 Vh
pI
+
+ - - - - - -
+ + + + + + + -
pH 4 pH 7
Cathode Anode
Place wet wicks (DH2O) under each end of strip
Set chiller temperature for 20degC (setting ~25)
Remove IPG strip from tray
Let oil drip off the strip
Place IPG strip gel facing up in equilibration tray
+ -
Add 10 ml equilibration buffer 1 per tray
+ -
+ -
+ -
+ -
(in chemical hood)
MW 4 7
+ -
10 DuracrylTM gel (22 cm x 23 cm x 1 mm)
bull TrisTricineSDS buffer
bull IPG strip
bull MW (gel worm)
bull Agarose overlay
bull Silicone spacer
bull Gasket
-
+
Conditions
bull Number of gel (1-10)
bull Max voltage 500 V
bull Max power 1600 mWgel
bull 4degC (setting 10)
bull ~19 hrs
10 DuracrylTM gel (22 cm x 23 cm x 1 mm)
MW 4 7
+ -
Trisacetate 25 mM Tris-base pH 83 (acetic acid)
200 mM Tris-base 200 mM Tricine 04 SDS
045 mm filtered
TrisTricine SDS
Bio-Rad
Digest to peptide fragment MS analysis
1 First dimension
isoelectric focusing
separation according to the pI
2 Second dimension
SDS electrophoresis (SDS-PAGE)
Separation according to the MW
Interested spot
Sensitivity Process
TimeSteps
Advantages
bullBio-safe Coomassie 10ng 25hr3 steps MS compatible easily visualised non
hazardous
bullCoomassie Blue R-250 40ng 25hr2 steps Oldest and least expensive method
bullSilver Stain Plus 1ng 15hr3 steps MS compatible high sensitivity low
background
bullBio-Rad silver stain 1ng 2hr7 steps High sensitivity detects some highly
glycosylated and other difficult to
stain proteins
bullSypro ruby 1ng 3hr2 steps MS compatible allows analysis in
flourescent imagers linear over 3
orders of magnitude
Coomassie staining
bull moderate sensitivity (36-47 ng)
bull non specific
bull not quantitative
Silver staining
bull sensitive (05-12 ng)
bull time consuming
bull non specific
bull negative stain some spots
Fluorescent dye (SYPRO ruby)
bull sensitive (1-2 ng)
bull specific quantitative
bull end point stain
Coomassie Silver
Coomassie Silver Stain Copper Stain
SDS-polyacrylamide gel
Electrotransfer to membrane
Phosphoimager for 32P and 35S labelled
1D or 2D gels
Fluoroimager for
SYPRO labelled
1D or 2D gels
Densitometer or
Photo Scanner
Normal liver
Tumor
Both
Melanie 4 (GeneBio - Windows only)
httpcaexpasyorgmelanie
ImageMaster 2D Elite (Amersham)
httpwwwimsupportcom
Phoretix 2D Advanced
httpwwwphoretixcom
PDQuest 61 (BioRad - Windows only) httpwwwproteomeworksbio-radcomhtmlpdquesthtml
Mass Spectrometry
wwwexpasych - Swiss-2DPAGE httpwwwanlgovBIOPMG - Mouse liver human breast cell lines pyrococcus Argonne Protein Mapping Group httpwwwharefieldnthamesnhsuknhliproteinindexhtml - HSC-2DPAGE Heart Science Centre Harefield Hospital httpotowustleduthcperi-gelshtm - Washington Univ Inner Ear Protein Database
httpcaexpasyorgch2d2d-indexhtml - World 2DPAGE Index of 2D gel databases
Time line ~ 4 days
bull Protein gt 250 kDa do not enter 2 SDS-PAGE properly
bull PH more than 9
bull PH less than 4
bull Hydrophobic proteins
bull Low abundant proteins
Immunoblotting Proteins from 2D gels were transferred to PVDFmembranes The membranes were blocked with 5 skimmed milk in PBS-Tween (PBS-T) for 3 h The membranes were incubated overnight at 4degC with individual sera diluted 1100 in PBS-T followed by secondary antibody (anti-human IgG conjugated with horseradish peroxidase) for 2 h with dilutions of 13000 Before and after addition of the secondary antibody membranes washed 4 times each time for 5 min in PBS-T To visualize antigenic components membranes were incubated in diaminobenzeden (DAB) and H2O2 for 15 min
List of protein spots that reacted with more than 30 of serum samples
and were identified by mass spectrometry
1Heat shock protein HSP 90-beta
2 precursor(GRP 78) 78 kDa glucose-regulated protein
3 Heat shock cognate 71 kDa protein
4 Stress-70 protein mitochondrial precursor
5 Tubulin beta-5 chain
6Tubulin alpha-6 chain and 60 kDa heat shock protein mitochondrial
precursor (Hsp60)
7 Heterogeneous nuclear ribonucleoprotein K (hnRNP K)
8 Cytokeratin 8
9 Cytokeratin 18
10 Beta-actin
11 cytokeratin 19
12 Eukaryotic initiation factor 4A-I
13 14-3-3 protein epsilon
14 14-3-3 protein zetadelta (Protein kinase Cinhibitor protein-1)
15 Translationally controlled tumor protein (TCTP)
Among these autoantigens cytokeratin 8 cytokeratin 18 Fructose-bisphosphate aldolase and enolase were reacted with more than 60 of healthy individualsrsquo serum samples These antigens were intense spots in gels
Heat Shock proteins tubulins heterogeneous nuclear ribonucleoprotein actins cytokeratins 14-3-3 protein epsilon 14-3-3 protein zetadelta eukaryotic initiation factors Rho GDP-dissociation inhibitors phosphoglycerate mutase 1 G6PD GA3PD peroxiredoxins F-acting capping protein and triosephosphate isomerase were previously reported to induce humoral arm of the immune response in cancer
Translationally controlled tumor protein and electron transfer flavoprotein alpha-subunit have not been reported to induce humoral arm of the immune response in cancer so far
In terms of biological processes the identified proteins
belong to several groups
They are mainely involved in cell motility cytoskleton
stress response apoptosis cell signaling and
metabolism
Identification of other immunogenic proteins is
underway
Appel RD et al 1993 - SWISS-2DPAGE a database of two-dimensional gel electrophoresis images Electrophoresis 14 1232-1238
Appel RD Bairoch A Sanchez JC Vargas JR Golaz O Pasquali C and Hochstrasser DF 1996 ndash Federated two-dimensional electrophoresis database a simple means of publishing two-dimensional electrophoresis data Electrophoresis 17 540-546
Bjellqvist B Ek K Righetti PG Gianazza E Gorg A Westermeier R Postel W 1982 ndash Isoelectric focusing in immobilised pH gradients principle methodology and applications JBiochemBiophysMethods 6 317-339
Brazma A et al 2001 ndash Minimum information about a microarray experiment (MIAME)-towards standards for microarray data Nat genetics 29 365-71
Hoogland C Baujard Sanchez JC Hochstrasser DF and Appel RD 1997 ndash Make2ddb a simple package to set up a two-diensional electrophoresis database for the world wide web Electrophoresis 18 2755-2758
OFarrell 1975 - High resolution two-dimensional electrophoresis of proteins JBiolChem 25 250 4007-21
2 Dimensional Electrophoresis
Proteomics is the large-scale study of proteins
particularly their structures and functions
The proteome is the entire complement of proteins produced by an organism or system This will vary with time
Proteomics is often considered the next step in the study of biological systems after genomics
Why study Proteomics
First the level of transcription of a gene gives only a rough estimate of its level of
expression into a protein An mRNA produced in abundance may be degraded rapidly
or translated inefficiently resulting in a small amount of protein
Second many proteins experience post-translational modifications that profoundly
affect their activities for example some proteins are not active until they become
phosphorylated Methods such as phosphoproteomics and glycoproteomics are used to
study post-translational modifications
Third many transcripts give rise to more than one protein through alternative splicing
or alternative post-translational modifications
Finally At present there is no other technique that is capable of simultaneously
resolving thousands of proteins in one separation procedure
The core technology of proteomics is 2-DE
To identify as many components of the proteome as possible
Protein Mining catalog all the proteins present in a tissue cell organelle etc
Protein Function
Mapping Protein Post-Translational Modification - Characterization of posttranslational modifications phosphorylation glycosylation oxidation etc
Protein Localization
Protein Expression Studies
Comparative proteomics
- proteomics monitors the expression of a large number of proteins within a cell or tissue and observers quantitatively how the
patte of expression changes under different circumstances (comparative proteomics) such as
bull Healthy Diseased
bull Cancerous Benign
bull Drug resistant Drug susceptible
bull Tissue specific
An interesting use of proteomics is using specific protein biomarkers to diagnose disease
Sample Preparation 2-D Electrophoresis Spot Detection amp Image Analysis
Enzymatic Digestion Peptide-Mass Fingerprinting
Peptide Sequencing via MS Database Search
Protein Identification
(I) (III)
(IV) (V) (VI)
(II)
Sample preparation buffer composition IPG strip rehydration 1st dimension IEF run IPG strip wash Equilibration buffer Equilibration buffer 2nd dimension SDS-PAGE Gel staining Immunoblot Imaging
Sample preparation
buffer composition
IPG strip rehydration
1st dimension IEF run
IPG strip wash
Equilibration buffer I
Equilibration buffer II
2nd dimension SDS-PAGE
Gel staining
Imaging
2-D Electrophoresis
Consistent protocol Limit sample degradation
bull fresh cellstissue bull protease inhibitors bull keep sample cold bull long term storage at ndash80degC
Limit sample contamination
bull gloves (keratin)
SolubilisationDenaturation buffer
bull separate proteins into individual components
bull reliable running in the IEF
Gentle lysis method 1 Osmotic lysis (cultured cells)
Suspend cells in hypoosmotic solution
2 Repeated freezing and thawing (bacteria)
Freeze using liquid nitrogen
3 Detergent lysis (yeast and fungi)
Lysis buffer (containing urea and detergent)
SDS (have to be removed before IEF)
4 Enzymatic lysis (plant bacteria fungi)
Lysomzyme (bacteria)
Cellulose and pectinase (plant)
Lyticase (yeast)
Vigorous lysis method
1 Sonication probe (cell suspension)
Avoid overheat cool on ice between burst
2 French pressure (microorganism with cell wall)
Cells are lysed by shear force
3 Mortar and pestle (solid tissue microorganism)
Grind solid tissue to fine powder with liquid nitrogen
4 Sample grinding kit (for small amount of sample)
For precious sample
5 Glass bead (cell suspension microorganism)
Using abrasive vortexed bead to break cell walls
Chaotrope
Urea (up to 9 M)
Thiourea (up to 2 M)
bull Disrupt of hydrogen and hydrophobic bonds
Note Urea (if tdeggt37degC) cyanate (HN=C=O)
carbamylation (Lys Arg)
(R-NH2 R-NH-CO-NH2)
carbamylation trains
Reductants
b-mercaptoethanol
DTT(dithiothreitol)
bull Break disulfide bridge
(within or between protein)
Detergents ndash surfactants
CHAPS (up to 4)
bull Disrupt membranes
bull Break hydrophobic interactions
bull Solubilise lipids
bull Release membrane bound proteins
Note No net charge for IEF (no SDS)
Soluble in urea
Isoelectric Focusing
Measure the protein concenteration in you samples
1 Biuret
2 Lowry methods
3 Bradford methods
4 UV methods
5 Special methods
6 Other commercial methods
BCA assay (bicinchoninic acid assay Pierce)
DC protein assay (detergent compatible Bio-rad)
DCRC protein assay (detergentreducing agent compatible
Bio-rad)
Before runninng IEF you shouldhellip
Introduced by Gorg A
Ref Gorg A (1994) Westermeier (2001)
Dried gel strips can be stored at -20 to -
80 from months to years
Amersham Biosciences Bio-Rad
IPG strips (3 mm x 18 cm x 05 mm)
Narrow range
Medium range
Broad range
4 7
35 45 55 67
40 50 60
3 10
6 11
Load 400 ml sample per groove (no bubbles)
Place IPG strip gel facing down in groove
Add ~25 ml non conductive oil per groove
Peel off protective film from strip
Rehydrate overnight (~22 hrs) at room temperature
Program power supply
bull Number of gels (1-10)
bull Max voltage 5000 V
bull Vhold 125 V
bull Duration 24 hrs 00 min
bull Max current 80 mAstrip
bull Volt hours 80000 Vh
pI
+
+ - - - - - -
+ + + + + + + -
pH 4 pH 7
Cathode Anode
Place wet wicks (DH2O) under each end of strip
Set chiller temperature for 20degC (setting ~25)
Remove IPG strip from tray
Let oil drip off the strip
Place IPG strip gel facing up in equilibration tray
+ -
Add 10 ml equilibration buffer 1 per tray
+ -
+ -
+ -
+ -
(in chemical hood)
MW 4 7
+ -
10 DuracrylTM gel (22 cm x 23 cm x 1 mm)
bull TrisTricineSDS buffer
bull IPG strip
bull MW (gel worm)
bull Agarose overlay
bull Silicone spacer
bull Gasket
-
+
Conditions
bull Number of gel (1-10)
bull Max voltage 500 V
bull Max power 1600 mWgel
bull 4degC (setting 10)
bull ~19 hrs
10 DuracrylTM gel (22 cm x 23 cm x 1 mm)
MW 4 7
+ -
Trisacetate 25 mM Tris-base pH 83 (acetic acid)
200 mM Tris-base 200 mM Tricine 04 SDS
045 mm filtered
TrisTricine SDS
Bio-Rad
Digest to peptide fragment MS analysis
1 First dimension
isoelectric focusing
separation according to the pI
2 Second dimension
SDS electrophoresis (SDS-PAGE)
Separation according to the MW
Interested spot
Sensitivity Process
TimeSteps
Advantages
bullBio-safe Coomassie 10ng 25hr3 steps MS compatible easily visualised non
hazardous
bullCoomassie Blue R-250 40ng 25hr2 steps Oldest and least expensive method
bullSilver Stain Plus 1ng 15hr3 steps MS compatible high sensitivity low
background
bullBio-Rad silver stain 1ng 2hr7 steps High sensitivity detects some highly
glycosylated and other difficult to
stain proteins
bullSypro ruby 1ng 3hr2 steps MS compatible allows analysis in
flourescent imagers linear over 3
orders of magnitude
Coomassie staining
bull moderate sensitivity (36-47 ng)
bull non specific
bull not quantitative
Silver staining
bull sensitive (05-12 ng)
bull time consuming
bull non specific
bull negative stain some spots
Fluorescent dye (SYPRO ruby)
bull sensitive (1-2 ng)
bull specific quantitative
bull end point stain
Coomassie Silver
Coomassie Silver Stain Copper Stain
SDS-polyacrylamide gel
Electrotransfer to membrane
Phosphoimager for 32P and 35S labelled
1D or 2D gels
Fluoroimager for
SYPRO labelled
1D or 2D gels
Densitometer or
Photo Scanner
Normal liver
Tumor
Both
Melanie 4 (GeneBio - Windows only)
httpcaexpasyorgmelanie
ImageMaster 2D Elite (Amersham)
httpwwwimsupportcom
Phoretix 2D Advanced
httpwwwphoretixcom
PDQuest 61 (BioRad - Windows only) httpwwwproteomeworksbio-radcomhtmlpdquesthtml
Mass Spectrometry
wwwexpasych - Swiss-2DPAGE httpwwwanlgovBIOPMG - Mouse liver human breast cell lines pyrococcus Argonne Protein Mapping Group httpwwwharefieldnthamesnhsuknhliproteinindexhtml - HSC-2DPAGE Heart Science Centre Harefield Hospital httpotowustleduthcperi-gelshtm - Washington Univ Inner Ear Protein Database
httpcaexpasyorgch2d2d-indexhtml - World 2DPAGE Index of 2D gel databases
Time line ~ 4 days
bull Protein gt 250 kDa do not enter 2 SDS-PAGE properly
bull PH more than 9
bull PH less than 4
bull Hydrophobic proteins
bull Low abundant proteins
Immunoblotting Proteins from 2D gels were transferred to PVDFmembranes The membranes were blocked with 5 skimmed milk in PBS-Tween (PBS-T) for 3 h The membranes were incubated overnight at 4degC with individual sera diluted 1100 in PBS-T followed by secondary antibody (anti-human IgG conjugated with horseradish peroxidase) for 2 h with dilutions of 13000 Before and after addition of the secondary antibody membranes washed 4 times each time for 5 min in PBS-T To visualize antigenic components membranes were incubated in diaminobenzeden (DAB) and H2O2 for 15 min
List of protein spots that reacted with more than 30 of serum samples
and were identified by mass spectrometry
1Heat shock protein HSP 90-beta
2 precursor(GRP 78) 78 kDa glucose-regulated protein
3 Heat shock cognate 71 kDa protein
4 Stress-70 protein mitochondrial precursor
5 Tubulin beta-5 chain
6Tubulin alpha-6 chain and 60 kDa heat shock protein mitochondrial
precursor (Hsp60)
7 Heterogeneous nuclear ribonucleoprotein K (hnRNP K)
8 Cytokeratin 8
9 Cytokeratin 18
10 Beta-actin
11 cytokeratin 19
12 Eukaryotic initiation factor 4A-I
13 14-3-3 protein epsilon
14 14-3-3 protein zetadelta (Protein kinase Cinhibitor protein-1)
15 Translationally controlled tumor protein (TCTP)
Among these autoantigens cytokeratin 8 cytokeratin 18 Fructose-bisphosphate aldolase and enolase were reacted with more than 60 of healthy individualsrsquo serum samples These antigens were intense spots in gels
Heat Shock proteins tubulins heterogeneous nuclear ribonucleoprotein actins cytokeratins 14-3-3 protein epsilon 14-3-3 protein zetadelta eukaryotic initiation factors Rho GDP-dissociation inhibitors phosphoglycerate mutase 1 G6PD GA3PD peroxiredoxins F-acting capping protein and triosephosphate isomerase were previously reported to induce humoral arm of the immune response in cancer
Translationally controlled tumor protein and electron transfer flavoprotein alpha-subunit have not been reported to induce humoral arm of the immune response in cancer so far
In terms of biological processes the identified proteins
belong to several groups
They are mainely involved in cell motility cytoskleton
stress response apoptosis cell signaling and
metabolism
Identification of other immunogenic proteins is
underway
Appel RD et al 1993 - SWISS-2DPAGE a database of two-dimensional gel electrophoresis images Electrophoresis 14 1232-1238
Appel RD Bairoch A Sanchez JC Vargas JR Golaz O Pasquali C and Hochstrasser DF 1996 ndash Federated two-dimensional electrophoresis database a simple means of publishing two-dimensional electrophoresis data Electrophoresis 17 540-546
Bjellqvist B Ek K Righetti PG Gianazza E Gorg A Westermeier R Postel W 1982 ndash Isoelectric focusing in immobilised pH gradients principle methodology and applications JBiochemBiophysMethods 6 317-339
Brazma A et al 2001 ndash Minimum information about a microarray experiment (MIAME)-towards standards for microarray data Nat genetics 29 365-71
Hoogland C Baujard Sanchez JC Hochstrasser DF and Appel RD 1997 ndash Make2ddb a simple package to set up a two-diensional electrophoresis database for the world wide web Electrophoresis 18 2755-2758
OFarrell 1975 - High resolution two-dimensional electrophoresis of proteins JBiolChem 25 250 4007-21
Proteomics is the large-scale study of proteins
particularly their structures and functions
The proteome is the entire complement of proteins produced by an organism or system This will vary with time
Proteomics is often considered the next step in the study of biological systems after genomics
Why study Proteomics
First the level of transcription of a gene gives only a rough estimate of its level of
expression into a protein An mRNA produced in abundance may be degraded rapidly
or translated inefficiently resulting in a small amount of protein
Second many proteins experience post-translational modifications that profoundly
affect their activities for example some proteins are not active until they become
phosphorylated Methods such as phosphoproteomics and glycoproteomics are used to
study post-translational modifications
Third many transcripts give rise to more than one protein through alternative splicing
or alternative post-translational modifications
Finally At present there is no other technique that is capable of simultaneously
resolving thousands of proteins in one separation procedure
The core technology of proteomics is 2-DE
To identify as many components of the proteome as possible
Protein Mining catalog all the proteins present in a tissue cell organelle etc
Protein Function
Mapping Protein Post-Translational Modification - Characterization of posttranslational modifications phosphorylation glycosylation oxidation etc
Protein Localization
Protein Expression Studies
Comparative proteomics
- proteomics monitors the expression of a large number of proteins within a cell or tissue and observers quantitatively how the
patte of expression changes under different circumstances (comparative proteomics) such as
bull Healthy Diseased
bull Cancerous Benign
bull Drug resistant Drug susceptible
bull Tissue specific
An interesting use of proteomics is using specific protein biomarkers to diagnose disease
Sample Preparation 2-D Electrophoresis Spot Detection amp Image Analysis
Enzymatic Digestion Peptide-Mass Fingerprinting
Peptide Sequencing via MS Database Search
Protein Identification
(I) (III)
(IV) (V) (VI)
(II)
Sample preparation buffer composition IPG strip rehydration 1st dimension IEF run IPG strip wash Equilibration buffer Equilibration buffer 2nd dimension SDS-PAGE Gel staining Immunoblot Imaging
Sample preparation
buffer composition
IPG strip rehydration
1st dimension IEF run
IPG strip wash
Equilibration buffer I
Equilibration buffer II
2nd dimension SDS-PAGE
Gel staining
Imaging
2-D Electrophoresis
Consistent protocol Limit sample degradation
bull fresh cellstissue bull protease inhibitors bull keep sample cold bull long term storage at ndash80degC
Limit sample contamination
bull gloves (keratin)
SolubilisationDenaturation buffer
bull separate proteins into individual components
bull reliable running in the IEF
Gentle lysis method 1 Osmotic lysis (cultured cells)
Suspend cells in hypoosmotic solution
2 Repeated freezing and thawing (bacteria)
Freeze using liquid nitrogen
3 Detergent lysis (yeast and fungi)
Lysis buffer (containing urea and detergent)
SDS (have to be removed before IEF)
4 Enzymatic lysis (plant bacteria fungi)
Lysomzyme (bacteria)
Cellulose and pectinase (plant)
Lyticase (yeast)
Vigorous lysis method
1 Sonication probe (cell suspension)
Avoid overheat cool on ice between burst
2 French pressure (microorganism with cell wall)
Cells are lysed by shear force
3 Mortar and pestle (solid tissue microorganism)
Grind solid tissue to fine powder with liquid nitrogen
4 Sample grinding kit (for small amount of sample)
For precious sample
5 Glass bead (cell suspension microorganism)
Using abrasive vortexed bead to break cell walls
Chaotrope
Urea (up to 9 M)
Thiourea (up to 2 M)
bull Disrupt of hydrogen and hydrophobic bonds
Note Urea (if tdeggt37degC) cyanate (HN=C=O)
carbamylation (Lys Arg)
(R-NH2 R-NH-CO-NH2)
carbamylation trains
Reductants
b-mercaptoethanol
DTT(dithiothreitol)
bull Break disulfide bridge
(within or between protein)
Detergents ndash surfactants
CHAPS (up to 4)
bull Disrupt membranes
bull Break hydrophobic interactions
bull Solubilise lipids
bull Release membrane bound proteins
Note No net charge for IEF (no SDS)
Soluble in urea
Isoelectric Focusing
Measure the protein concenteration in you samples
1 Biuret
2 Lowry methods
3 Bradford methods
4 UV methods
5 Special methods
6 Other commercial methods
BCA assay (bicinchoninic acid assay Pierce)
DC protein assay (detergent compatible Bio-rad)
DCRC protein assay (detergentreducing agent compatible
Bio-rad)
Before runninng IEF you shouldhellip
Introduced by Gorg A
Ref Gorg A (1994) Westermeier (2001)
Dried gel strips can be stored at -20 to -
80 from months to years
Amersham Biosciences Bio-Rad
IPG strips (3 mm x 18 cm x 05 mm)
Narrow range
Medium range
Broad range
4 7
35 45 55 67
40 50 60
3 10
6 11
Load 400 ml sample per groove (no bubbles)
Place IPG strip gel facing down in groove
Add ~25 ml non conductive oil per groove
Peel off protective film from strip
Rehydrate overnight (~22 hrs) at room temperature
Program power supply
bull Number of gels (1-10)
bull Max voltage 5000 V
bull Vhold 125 V
bull Duration 24 hrs 00 min
bull Max current 80 mAstrip
bull Volt hours 80000 Vh
pI
+
+ - - - - - -
+ + + + + + + -
pH 4 pH 7
Cathode Anode
Place wet wicks (DH2O) under each end of strip
Set chiller temperature for 20degC (setting ~25)
Remove IPG strip from tray
Let oil drip off the strip
Place IPG strip gel facing up in equilibration tray
+ -
Add 10 ml equilibration buffer 1 per tray
+ -
+ -
+ -
+ -
(in chemical hood)
MW 4 7
+ -
10 DuracrylTM gel (22 cm x 23 cm x 1 mm)
bull TrisTricineSDS buffer
bull IPG strip
bull MW (gel worm)
bull Agarose overlay
bull Silicone spacer
bull Gasket
-
+
Conditions
bull Number of gel (1-10)
bull Max voltage 500 V
bull Max power 1600 mWgel
bull 4degC (setting 10)
bull ~19 hrs
10 DuracrylTM gel (22 cm x 23 cm x 1 mm)
MW 4 7
+ -
Trisacetate 25 mM Tris-base pH 83 (acetic acid)
200 mM Tris-base 200 mM Tricine 04 SDS
045 mm filtered
TrisTricine SDS
Bio-Rad
Digest to peptide fragment MS analysis
1 First dimension
isoelectric focusing
separation according to the pI
2 Second dimension
SDS electrophoresis (SDS-PAGE)
Separation according to the MW
Interested spot
Sensitivity Process
TimeSteps
Advantages
bullBio-safe Coomassie 10ng 25hr3 steps MS compatible easily visualised non
hazardous
bullCoomassie Blue R-250 40ng 25hr2 steps Oldest and least expensive method
bullSilver Stain Plus 1ng 15hr3 steps MS compatible high sensitivity low
background
bullBio-Rad silver stain 1ng 2hr7 steps High sensitivity detects some highly
glycosylated and other difficult to
stain proteins
bullSypro ruby 1ng 3hr2 steps MS compatible allows analysis in
flourescent imagers linear over 3
orders of magnitude
Coomassie staining
bull moderate sensitivity (36-47 ng)
bull non specific
bull not quantitative
Silver staining
bull sensitive (05-12 ng)
bull time consuming
bull non specific
bull negative stain some spots
Fluorescent dye (SYPRO ruby)
bull sensitive (1-2 ng)
bull specific quantitative
bull end point stain
Coomassie Silver
Coomassie Silver Stain Copper Stain
SDS-polyacrylamide gel
Electrotransfer to membrane
Phosphoimager for 32P and 35S labelled
1D or 2D gels
Fluoroimager for
SYPRO labelled
1D or 2D gels
Densitometer or
Photo Scanner
Normal liver
Tumor
Both
Melanie 4 (GeneBio - Windows only)
httpcaexpasyorgmelanie
ImageMaster 2D Elite (Amersham)
httpwwwimsupportcom
Phoretix 2D Advanced
httpwwwphoretixcom
PDQuest 61 (BioRad - Windows only) httpwwwproteomeworksbio-radcomhtmlpdquesthtml
Mass Spectrometry
wwwexpasych - Swiss-2DPAGE httpwwwanlgovBIOPMG - Mouse liver human breast cell lines pyrococcus Argonne Protein Mapping Group httpwwwharefieldnthamesnhsuknhliproteinindexhtml - HSC-2DPAGE Heart Science Centre Harefield Hospital httpotowustleduthcperi-gelshtm - Washington Univ Inner Ear Protein Database
httpcaexpasyorgch2d2d-indexhtml - World 2DPAGE Index of 2D gel databases
Time line ~ 4 days
bull Protein gt 250 kDa do not enter 2 SDS-PAGE properly
bull PH more than 9
bull PH less than 4
bull Hydrophobic proteins
bull Low abundant proteins
Immunoblotting Proteins from 2D gels were transferred to PVDFmembranes The membranes were blocked with 5 skimmed milk in PBS-Tween (PBS-T) for 3 h The membranes were incubated overnight at 4degC with individual sera diluted 1100 in PBS-T followed by secondary antibody (anti-human IgG conjugated with horseradish peroxidase) for 2 h with dilutions of 13000 Before and after addition of the secondary antibody membranes washed 4 times each time for 5 min in PBS-T To visualize antigenic components membranes were incubated in diaminobenzeden (DAB) and H2O2 for 15 min
List of protein spots that reacted with more than 30 of serum samples
and were identified by mass spectrometry
1Heat shock protein HSP 90-beta
2 precursor(GRP 78) 78 kDa glucose-regulated protein
3 Heat shock cognate 71 kDa protein
4 Stress-70 protein mitochondrial precursor
5 Tubulin beta-5 chain
6Tubulin alpha-6 chain and 60 kDa heat shock protein mitochondrial
precursor (Hsp60)
7 Heterogeneous nuclear ribonucleoprotein K (hnRNP K)
8 Cytokeratin 8
9 Cytokeratin 18
10 Beta-actin
11 cytokeratin 19
12 Eukaryotic initiation factor 4A-I
13 14-3-3 protein epsilon
14 14-3-3 protein zetadelta (Protein kinase Cinhibitor protein-1)
15 Translationally controlled tumor protein (TCTP)
Among these autoantigens cytokeratin 8 cytokeratin 18 Fructose-bisphosphate aldolase and enolase were reacted with more than 60 of healthy individualsrsquo serum samples These antigens were intense spots in gels
Heat Shock proteins tubulins heterogeneous nuclear ribonucleoprotein actins cytokeratins 14-3-3 protein epsilon 14-3-3 protein zetadelta eukaryotic initiation factors Rho GDP-dissociation inhibitors phosphoglycerate mutase 1 G6PD GA3PD peroxiredoxins F-acting capping protein and triosephosphate isomerase were previously reported to induce humoral arm of the immune response in cancer
Translationally controlled tumor protein and electron transfer flavoprotein alpha-subunit have not been reported to induce humoral arm of the immune response in cancer so far
In terms of biological processes the identified proteins
belong to several groups
They are mainely involved in cell motility cytoskleton
stress response apoptosis cell signaling and
metabolism
Identification of other immunogenic proteins is
underway
Appel RD et al 1993 - SWISS-2DPAGE a database of two-dimensional gel electrophoresis images Electrophoresis 14 1232-1238
Appel RD Bairoch A Sanchez JC Vargas JR Golaz O Pasquali C and Hochstrasser DF 1996 ndash Federated two-dimensional electrophoresis database a simple means of publishing two-dimensional electrophoresis data Electrophoresis 17 540-546
Bjellqvist B Ek K Righetti PG Gianazza E Gorg A Westermeier R Postel W 1982 ndash Isoelectric focusing in immobilised pH gradients principle methodology and applications JBiochemBiophysMethods 6 317-339
Brazma A et al 2001 ndash Minimum information about a microarray experiment (MIAME)-towards standards for microarray data Nat genetics 29 365-71
Hoogland C Baujard Sanchez JC Hochstrasser DF and Appel RD 1997 ndash Make2ddb a simple package to set up a two-diensional electrophoresis database for the world wide web Electrophoresis 18 2755-2758
OFarrell 1975 - High resolution two-dimensional electrophoresis of proteins JBiolChem 25 250 4007-21
Why study Proteomics
First the level of transcription of a gene gives only a rough estimate of its level of
expression into a protein An mRNA produced in abundance may be degraded rapidly
or translated inefficiently resulting in a small amount of protein
Second many proteins experience post-translational modifications that profoundly
affect their activities for example some proteins are not active until they become
phosphorylated Methods such as phosphoproteomics and glycoproteomics are used to
study post-translational modifications
Third many transcripts give rise to more than one protein through alternative splicing
or alternative post-translational modifications
Finally At present there is no other technique that is capable of simultaneously
resolving thousands of proteins in one separation procedure
The core technology of proteomics is 2-DE
To identify as many components of the proteome as possible
Protein Mining catalog all the proteins present in a tissue cell organelle etc
Protein Function
Mapping Protein Post-Translational Modification - Characterization of posttranslational modifications phosphorylation glycosylation oxidation etc
Protein Localization
Protein Expression Studies
Comparative proteomics
- proteomics monitors the expression of a large number of proteins within a cell or tissue and observers quantitatively how the
patte of expression changes under different circumstances (comparative proteomics) such as
bull Healthy Diseased
bull Cancerous Benign
bull Drug resistant Drug susceptible
bull Tissue specific
An interesting use of proteomics is using specific protein biomarkers to diagnose disease
Sample Preparation 2-D Electrophoresis Spot Detection amp Image Analysis
Enzymatic Digestion Peptide-Mass Fingerprinting
Peptide Sequencing via MS Database Search
Protein Identification
(I) (III)
(IV) (V) (VI)
(II)
Sample preparation buffer composition IPG strip rehydration 1st dimension IEF run IPG strip wash Equilibration buffer Equilibration buffer 2nd dimension SDS-PAGE Gel staining Immunoblot Imaging
Sample preparation
buffer composition
IPG strip rehydration
1st dimension IEF run
IPG strip wash
Equilibration buffer I
Equilibration buffer II
2nd dimension SDS-PAGE
Gel staining
Imaging
2-D Electrophoresis
Consistent protocol Limit sample degradation
bull fresh cellstissue bull protease inhibitors bull keep sample cold bull long term storage at ndash80degC
Limit sample contamination
bull gloves (keratin)
SolubilisationDenaturation buffer
bull separate proteins into individual components
bull reliable running in the IEF
Gentle lysis method 1 Osmotic lysis (cultured cells)
Suspend cells in hypoosmotic solution
2 Repeated freezing and thawing (bacteria)
Freeze using liquid nitrogen
3 Detergent lysis (yeast and fungi)
Lysis buffer (containing urea and detergent)
SDS (have to be removed before IEF)
4 Enzymatic lysis (plant bacteria fungi)
Lysomzyme (bacteria)
Cellulose and pectinase (plant)
Lyticase (yeast)
Vigorous lysis method
1 Sonication probe (cell suspension)
Avoid overheat cool on ice between burst
2 French pressure (microorganism with cell wall)
Cells are lysed by shear force
3 Mortar and pestle (solid tissue microorganism)
Grind solid tissue to fine powder with liquid nitrogen
4 Sample grinding kit (for small amount of sample)
For precious sample
5 Glass bead (cell suspension microorganism)
Using abrasive vortexed bead to break cell walls
Chaotrope
Urea (up to 9 M)
Thiourea (up to 2 M)
bull Disrupt of hydrogen and hydrophobic bonds
Note Urea (if tdeggt37degC) cyanate (HN=C=O)
carbamylation (Lys Arg)
(R-NH2 R-NH-CO-NH2)
carbamylation trains
Reductants
b-mercaptoethanol
DTT(dithiothreitol)
bull Break disulfide bridge
(within or between protein)
Detergents ndash surfactants
CHAPS (up to 4)
bull Disrupt membranes
bull Break hydrophobic interactions
bull Solubilise lipids
bull Release membrane bound proteins
Note No net charge for IEF (no SDS)
Soluble in urea
Isoelectric Focusing
Measure the protein concenteration in you samples
1 Biuret
2 Lowry methods
3 Bradford methods
4 UV methods
5 Special methods
6 Other commercial methods
BCA assay (bicinchoninic acid assay Pierce)
DC protein assay (detergent compatible Bio-rad)
DCRC protein assay (detergentreducing agent compatible
Bio-rad)
Before runninng IEF you shouldhellip
Introduced by Gorg A
Ref Gorg A (1994) Westermeier (2001)
Dried gel strips can be stored at -20 to -
80 from months to years
Amersham Biosciences Bio-Rad
IPG strips (3 mm x 18 cm x 05 mm)
Narrow range
Medium range
Broad range
4 7
35 45 55 67
40 50 60
3 10
6 11
Load 400 ml sample per groove (no bubbles)
Place IPG strip gel facing down in groove
Add ~25 ml non conductive oil per groove
Peel off protective film from strip
Rehydrate overnight (~22 hrs) at room temperature
Program power supply
bull Number of gels (1-10)
bull Max voltage 5000 V
bull Vhold 125 V
bull Duration 24 hrs 00 min
bull Max current 80 mAstrip
bull Volt hours 80000 Vh
pI
+
+ - - - - - -
+ + + + + + + -
pH 4 pH 7
Cathode Anode
Place wet wicks (DH2O) under each end of strip
Set chiller temperature for 20degC (setting ~25)
Remove IPG strip from tray
Let oil drip off the strip
Place IPG strip gel facing up in equilibration tray
+ -
Add 10 ml equilibration buffer 1 per tray
+ -
+ -
+ -
+ -
(in chemical hood)
MW 4 7
+ -
10 DuracrylTM gel (22 cm x 23 cm x 1 mm)
bull TrisTricineSDS buffer
bull IPG strip
bull MW (gel worm)
bull Agarose overlay
bull Silicone spacer
bull Gasket
-
+
Conditions
bull Number of gel (1-10)
bull Max voltage 500 V
bull Max power 1600 mWgel
bull 4degC (setting 10)
bull ~19 hrs
10 DuracrylTM gel (22 cm x 23 cm x 1 mm)
MW 4 7
+ -
Trisacetate 25 mM Tris-base pH 83 (acetic acid)
200 mM Tris-base 200 mM Tricine 04 SDS
045 mm filtered
TrisTricine SDS
Bio-Rad
Digest to peptide fragment MS analysis
1 First dimension
isoelectric focusing
separation according to the pI
2 Second dimension
SDS electrophoresis (SDS-PAGE)
Separation according to the MW
Interested spot
Sensitivity Process
TimeSteps
Advantages
bullBio-safe Coomassie 10ng 25hr3 steps MS compatible easily visualised non
hazardous
bullCoomassie Blue R-250 40ng 25hr2 steps Oldest and least expensive method
bullSilver Stain Plus 1ng 15hr3 steps MS compatible high sensitivity low
background
bullBio-Rad silver stain 1ng 2hr7 steps High sensitivity detects some highly
glycosylated and other difficult to
stain proteins
bullSypro ruby 1ng 3hr2 steps MS compatible allows analysis in
flourescent imagers linear over 3
orders of magnitude
Coomassie staining
bull moderate sensitivity (36-47 ng)
bull non specific
bull not quantitative
Silver staining
bull sensitive (05-12 ng)
bull time consuming
bull non specific
bull negative stain some spots
Fluorescent dye (SYPRO ruby)
bull sensitive (1-2 ng)
bull specific quantitative
bull end point stain
Coomassie Silver
Coomassie Silver Stain Copper Stain
SDS-polyacrylamide gel
Electrotransfer to membrane
Phosphoimager for 32P and 35S labelled
1D or 2D gels
Fluoroimager for
SYPRO labelled
1D or 2D gels
Densitometer or
Photo Scanner
Normal liver
Tumor
Both
Melanie 4 (GeneBio - Windows only)
httpcaexpasyorgmelanie
ImageMaster 2D Elite (Amersham)
httpwwwimsupportcom
Phoretix 2D Advanced
httpwwwphoretixcom
PDQuest 61 (BioRad - Windows only) httpwwwproteomeworksbio-radcomhtmlpdquesthtml
Mass Spectrometry
wwwexpasych - Swiss-2DPAGE httpwwwanlgovBIOPMG - Mouse liver human breast cell lines pyrococcus Argonne Protein Mapping Group httpwwwharefieldnthamesnhsuknhliproteinindexhtml - HSC-2DPAGE Heart Science Centre Harefield Hospital httpotowustleduthcperi-gelshtm - Washington Univ Inner Ear Protein Database
httpcaexpasyorgch2d2d-indexhtml - World 2DPAGE Index of 2D gel databases
Time line ~ 4 days
bull Protein gt 250 kDa do not enter 2 SDS-PAGE properly
bull PH more than 9
bull PH less than 4
bull Hydrophobic proteins
bull Low abundant proteins
Immunoblotting Proteins from 2D gels were transferred to PVDFmembranes The membranes were blocked with 5 skimmed milk in PBS-Tween (PBS-T) for 3 h The membranes were incubated overnight at 4degC with individual sera diluted 1100 in PBS-T followed by secondary antibody (anti-human IgG conjugated with horseradish peroxidase) for 2 h with dilutions of 13000 Before and after addition of the secondary antibody membranes washed 4 times each time for 5 min in PBS-T To visualize antigenic components membranes were incubated in diaminobenzeden (DAB) and H2O2 for 15 min
List of protein spots that reacted with more than 30 of serum samples
and were identified by mass spectrometry
1Heat shock protein HSP 90-beta
2 precursor(GRP 78) 78 kDa glucose-regulated protein
3 Heat shock cognate 71 kDa protein
4 Stress-70 protein mitochondrial precursor
5 Tubulin beta-5 chain
6Tubulin alpha-6 chain and 60 kDa heat shock protein mitochondrial
precursor (Hsp60)
7 Heterogeneous nuclear ribonucleoprotein K (hnRNP K)
8 Cytokeratin 8
9 Cytokeratin 18
10 Beta-actin
11 cytokeratin 19
12 Eukaryotic initiation factor 4A-I
13 14-3-3 protein epsilon
14 14-3-3 protein zetadelta (Protein kinase Cinhibitor protein-1)
15 Translationally controlled tumor protein (TCTP)
Among these autoantigens cytokeratin 8 cytokeratin 18 Fructose-bisphosphate aldolase and enolase were reacted with more than 60 of healthy individualsrsquo serum samples These antigens were intense spots in gels
Heat Shock proteins tubulins heterogeneous nuclear ribonucleoprotein actins cytokeratins 14-3-3 protein epsilon 14-3-3 protein zetadelta eukaryotic initiation factors Rho GDP-dissociation inhibitors phosphoglycerate mutase 1 G6PD GA3PD peroxiredoxins F-acting capping protein and triosephosphate isomerase were previously reported to induce humoral arm of the immune response in cancer
Translationally controlled tumor protein and electron transfer flavoprotein alpha-subunit have not been reported to induce humoral arm of the immune response in cancer so far
In terms of biological processes the identified proteins
belong to several groups
They are mainely involved in cell motility cytoskleton
stress response apoptosis cell signaling and
metabolism
Identification of other immunogenic proteins is
underway
Appel RD et al 1993 - SWISS-2DPAGE a database of two-dimensional gel electrophoresis images Electrophoresis 14 1232-1238
Appel RD Bairoch A Sanchez JC Vargas JR Golaz O Pasquali C and Hochstrasser DF 1996 ndash Federated two-dimensional electrophoresis database a simple means of publishing two-dimensional electrophoresis data Electrophoresis 17 540-546
Bjellqvist B Ek K Righetti PG Gianazza E Gorg A Westermeier R Postel W 1982 ndash Isoelectric focusing in immobilised pH gradients principle methodology and applications JBiochemBiophysMethods 6 317-339
Brazma A et al 2001 ndash Minimum information about a microarray experiment (MIAME)-towards standards for microarray data Nat genetics 29 365-71
Hoogland C Baujard Sanchez JC Hochstrasser DF and Appel RD 1997 ndash Make2ddb a simple package to set up a two-diensional electrophoresis database for the world wide web Electrophoresis 18 2755-2758
OFarrell 1975 - High resolution two-dimensional electrophoresis of proteins JBiolChem 25 250 4007-21
To identify as many components of the proteome as possible
Protein Mining catalog all the proteins present in a tissue cell organelle etc
Protein Function
Mapping Protein Post-Translational Modification - Characterization of posttranslational modifications phosphorylation glycosylation oxidation etc
Protein Localization
Protein Expression Studies
Comparative proteomics
- proteomics monitors the expression of a large number of proteins within a cell or tissue and observers quantitatively how the
patte of expression changes under different circumstances (comparative proteomics) such as
bull Healthy Diseased
bull Cancerous Benign
bull Drug resistant Drug susceptible
bull Tissue specific
An interesting use of proteomics is using specific protein biomarkers to diagnose disease
Sample Preparation 2-D Electrophoresis Spot Detection amp Image Analysis
Enzymatic Digestion Peptide-Mass Fingerprinting
Peptide Sequencing via MS Database Search
Protein Identification
(I) (III)
(IV) (V) (VI)
(II)
Sample preparation buffer composition IPG strip rehydration 1st dimension IEF run IPG strip wash Equilibration buffer Equilibration buffer 2nd dimension SDS-PAGE Gel staining Immunoblot Imaging
Sample preparation
buffer composition
IPG strip rehydration
1st dimension IEF run
IPG strip wash
Equilibration buffer I
Equilibration buffer II
2nd dimension SDS-PAGE
Gel staining
Imaging
2-D Electrophoresis
Consistent protocol Limit sample degradation
bull fresh cellstissue bull protease inhibitors bull keep sample cold bull long term storage at ndash80degC
Limit sample contamination
bull gloves (keratin)
SolubilisationDenaturation buffer
bull separate proteins into individual components
bull reliable running in the IEF
Gentle lysis method 1 Osmotic lysis (cultured cells)
Suspend cells in hypoosmotic solution
2 Repeated freezing and thawing (bacteria)
Freeze using liquid nitrogen
3 Detergent lysis (yeast and fungi)
Lysis buffer (containing urea and detergent)
SDS (have to be removed before IEF)
4 Enzymatic lysis (plant bacteria fungi)
Lysomzyme (bacteria)
Cellulose and pectinase (plant)
Lyticase (yeast)
Vigorous lysis method
1 Sonication probe (cell suspension)
Avoid overheat cool on ice between burst
2 French pressure (microorganism with cell wall)
Cells are lysed by shear force
3 Mortar and pestle (solid tissue microorganism)
Grind solid tissue to fine powder with liquid nitrogen
4 Sample grinding kit (for small amount of sample)
For precious sample
5 Glass bead (cell suspension microorganism)
Using abrasive vortexed bead to break cell walls
Chaotrope
Urea (up to 9 M)
Thiourea (up to 2 M)
bull Disrupt of hydrogen and hydrophobic bonds
Note Urea (if tdeggt37degC) cyanate (HN=C=O)
carbamylation (Lys Arg)
(R-NH2 R-NH-CO-NH2)
carbamylation trains
Reductants
b-mercaptoethanol
DTT(dithiothreitol)
bull Break disulfide bridge
(within or between protein)
Detergents ndash surfactants
CHAPS (up to 4)
bull Disrupt membranes
bull Break hydrophobic interactions
bull Solubilise lipids
bull Release membrane bound proteins
Note No net charge for IEF (no SDS)
Soluble in urea
Isoelectric Focusing
Measure the protein concenteration in you samples
1 Biuret
2 Lowry methods
3 Bradford methods
4 UV methods
5 Special methods
6 Other commercial methods
BCA assay (bicinchoninic acid assay Pierce)
DC protein assay (detergent compatible Bio-rad)
DCRC protein assay (detergentreducing agent compatible
Bio-rad)
Before runninng IEF you shouldhellip
Introduced by Gorg A
Ref Gorg A (1994) Westermeier (2001)
Dried gel strips can be stored at -20 to -
80 from months to years
Amersham Biosciences Bio-Rad
IPG strips (3 mm x 18 cm x 05 mm)
Narrow range
Medium range
Broad range
4 7
35 45 55 67
40 50 60
3 10
6 11
Load 400 ml sample per groove (no bubbles)
Place IPG strip gel facing down in groove
Add ~25 ml non conductive oil per groove
Peel off protective film from strip
Rehydrate overnight (~22 hrs) at room temperature
Program power supply
bull Number of gels (1-10)
bull Max voltage 5000 V
bull Vhold 125 V
bull Duration 24 hrs 00 min
bull Max current 80 mAstrip
bull Volt hours 80000 Vh
pI
+
+ - - - - - -
+ + + + + + + -
pH 4 pH 7
Cathode Anode
Place wet wicks (DH2O) under each end of strip
Set chiller temperature for 20degC (setting ~25)
Remove IPG strip from tray
Let oil drip off the strip
Place IPG strip gel facing up in equilibration tray
+ -
Add 10 ml equilibration buffer 1 per tray
+ -
+ -
+ -
+ -
(in chemical hood)
MW 4 7
+ -
10 DuracrylTM gel (22 cm x 23 cm x 1 mm)
bull TrisTricineSDS buffer
bull IPG strip
bull MW (gel worm)
bull Agarose overlay
bull Silicone spacer
bull Gasket
-
+
Conditions
bull Number of gel (1-10)
bull Max voltage 500 V
bull Max power 1600 mWgel
bull 4degC (setting 10)
bull ~19 hrs
10 DuracrylTM gel (22 cm x 23 cm x 1 mm)
MW 4 7
+ -
Trisacetate 25 mM Tris-base pH 83 (acetic acid)
200 mM Tris-base 200 mM Tricine 04 SDS
045 mm filtered
TrisTricine SDS
Bio-Rad
Digest to peptide fragment MS analysis
1 First dimension
isoelectric focusing
separation according to the pI
2 Second dimension
SDS electrophoresis (SDS-PAGE)
Separation according to the MW
Interested spot
Sensitivity Process
TimeSteps
Advantages
bullBio-safe Coomassie 10ng 25hr3 steps MS compatible easily visualised non
hazardous
bullCoomassie Blue R-250 40ng 25hr2 steps Oldest and least expensive method
bullSilver Stain Plus 1ng 15hr3 steps MS compatible high sensitivity low
background
bullBio-Rad silver stain 1ng 2hr7 steps High sensitivity detects some highly
glycosylated and other difficult to
stain proteins
bullSypro ruby 1ng 3hr2 steps MS compatible allows analysis in
flourescent imagers linear over 3
orders of magnitude
Coomassie staining
bull moderate sensitivity (36-47 ng)
bull non specific
bull not quantitative
Silver staining
bull sensitive (05-12 ng)
bull time consuming
bull non specific
bull negative stain some spots
Fluorescent dye (SYPRO ruby)
bull sensitive (1-2 ng)
bull specific quantitative
bull end point stain
Coomassie Silver
Coomassie Silver Stain Copper Stain
SDS-polyacrylamide gel
Electrotransfer to membrane
Phosphoimager for 32P and 35S labelled
1D or 2D gels
Fluoroimager for
SYPRO labelled
1D or 2D gels
Densitometer or
Photo Scanner
Normal liver
Tumor
Both
Melanie 4 (GeneBio - Windows only)
httpcaexpasyorgmelanie
ImageMaster 2D Elite (Amersham)
httpwwwimsupportcom
Phoretix 2D Advanced
httpwwwphoretixcom
PDQuest 61 (BioRad - Windows only) httpwwwproteomeworksbio-radcomhtmlpdquesthtml
Mass Spectrometry
wwwexpasych - Swiss-2DPAGE httpwwwanlgovBIOPMG - Mouse liver human breast cell lines pyrococcus Argonne Protein Mapping Group httpwwwharefieldnthamesnhsuknhliproteinindexhtml - HSC-2DPAGE Heart Science Centre Harefield Hospital httpotowustleduthcperi-gelshtm - Washington Univ Inner Ear Protein Database
httpcaexpasyorgch2d2d-indexhtml - World 2DPAGE Index of 2D gel databases
Time line ~ 4 days
bull Protein gt 250 kDa do not enter 2 SDS-PAGE properly
bull PH more than 9
bull PH less than 4
bull Hydrophobic proteins
bull Low abundant proteins
Immunoblotting Proteins from 2D gels were transferred to PVDFmembranes The membranes were blocked with 5 skimmed milk in PBS-Tween (PBS-T) for 3 h The membranes were incubated overnight at 4degC with individual sera diluted 1100 in PBS-T followed by secondary antibody (anti-human IgG conjugated with horseradish peroxidase) for 2 h with dilutions of 13000 Before and after addition of the secondary antibody membranes washed 4 times each time for 5 min in PBS-T To visualize antigenic components membranes were incubated in diaminobenzeden (DAB) and H2O2 for 15 min
List of protein spots that reacted with more than 30 of serum samples
and were identified by mass spectrometry
1Heat shock protein HSP 90-beta
2 precursor(GRP 78) 78 kDa glucose-regulated protein
3 Heat shock cognate 71 kDa protein
4 Stress-70 protein mitochondrial precursor
5 Tubulin beta-5 chain
6Tubulin alpha-6 chain and 60 kDa heat shock protein mitochondrial
precursor (Hsp60)
7 Heterogeneous nuclear ribonucleoprotein K (hnRNP K)
8 Cytokeratin 8
9 Cytokeratin 18
10 Beta-actin
11 cytokeratin 19
12 Eukaryotic initiation factor 4A-I
13 14-3-3 protein epsilon
14 14-3-3 protein zetadelta (Protein kinase Cinhibitor protein-1)
15 Translationally controlled tumor protein (TCTP)
Among these autoantigens cytokeratin 8 cytokeratin 18 Fructose-bisphosphate aldolase and enolase were reacted with more than 60 of healthy individualsrsquo serum samples These antigens were intense spots in gels
Heat Shock proteins tubulins heterogeneous nuclear ribonucleoprotein actins cytokeratins 14-3-3 protein epsilon 14-3-3 protein zetadelta eukaryotic initiation factors Rho GDP-dissociation inhibitors phosphoglycerate mutase 1 G6PD GA3PD peroxiredoxins F-acting capping protein and triosephosphate isomerase were previously reported to induce humoral arm of the immune response in cancer
Translationally controlled tumor protein and electron transfer flavoprotein alpha-subunit have not been reported to induce humoral arm of the immune response in cancer so far
In terms of biological processes the identified proteins
belong to several groups
They are mainely involved in cell motility cytoskleton
stress response apoptosis cell signaling and
metabolism
Identification of other immunogenic proteins is
underway
Appel RD et al 1993 - SWISS-2DPAGE a database of two-dimensional gel electrophoresis images Electrophoresis 14 1232-1238
Appel RD Bairoch A Sanchez JC Vargas JR Golaz O Pasquali C and Hochstrasser DF 1996 ndash Federated two-dimensional electrophoresis database a simple means of publishing two-dimensional electrophoresis data Electrophoresis 17 540-546
Bjellqvist B Ek K Righetti PG Gianazza E Gorg A Westermeier R Postel W 1982 ndash Isoelectric focusing in immobilised pH gradients principle methodology and applications JBiochemBiophysMethods 6 317-339
Brazma A et al 2001 ndash Minimum information about a microarray experiment (MIAME)-towards standards for microarray data Nat genetics 29 365-71
Hoogland C Baujard Sanchez JC Hochstrasser DF and Appel RD 1997 ndash Make2ddb a simple package to set up a two-diensional electrophoresis database for the world wide web Electrophoresis 18 2755-2758
OFarrell 1975 - High resolution two-dimensional electrophoresis of proteins JBiolChem 25 250 4007-21
Protein Mining catalog all the proteins present in a tissue cell organelle etc
Protein Function
Mapping Protein Post-Translational Modification - Characterization of posttranslational modifications phosphorylation glycosylation oxidation etc
Protein Localization
Protein Expression Studies
Comparative proteomics
- proteomics monitors the expression of a large number of proteins within a cell or tissue and observers quantitatively how the
patte of expression changes under different circumstances (comparative proteomics) such as
bull Healthy Diseased
bull Cancerous Benign
bull Drug resistant Drug susceptible
bull Tissue specific
An interesting use of proteomics is using specific protein biomarkers to diagnose disease
Sample Preparation 2-D Electrophoresis Spot Detection amp Image Analysis
Enzymatic Digestion Peptide-Mass Fingerprinting
Peptide Sequencing via MS Database Search
Protein Identification
(I) (III)
(IV) (V) (VI)
(II)
Sample preparation buffer composition IPG strip rehydration 1st dimension IEF run IPG strip wash Equilibration buffer Equilibration buffer 2nd dimension SDS-PAGE Gel staining Immunoblot Imaging
Sample preparation
buffer composition
IPG strip rehydration
1st dimension IEF run
IPG strip wash
Equilibration buffer I
Equilibration buffer II
2nd dimension SDS-PAGE
Gel staining
Imaging
2-D Electrophoresis
Consistent protocol Limit sample degradation
bull fresh cellstissue bull protease inhibitors bull keep sample cold bull long term storage at ndash80degC
Limit sample contamination
bull gloves (keratin)
SolubilisationDenaturation buffer
bull separate proteins into individual components
bull reliable running in the IEF
Gentle lysis method 1 Osmotic lysis (cultured cells)
Suspend cells in hypoosmotic solution
2 Repeated freezing and thawing (bacteria)
Freeze using liquid nitrogen
3 Detergent lysis (yeast and fungi)
Lysis buffer (containing urea and detergent)
SDS (have to be removed before IEF)
4 Enzymatic lysis (plant bacteria fungi)
Lysomzyme (bacteria)
Cellulose and pectinase (plant)
Lyticase (yeast)
Vigorous lysis method
1 Sonication probe (cell suspension)
Avoid overheat cool on ice between burst
2 French pressure (microorganism with cell wall)
Cells are lysed by shear force
3 Mortar and pestle (solid tissue microorganism)
Grind solid tissue to fine powder with liquid nitrogen
4 Sample grinding kit (for small amount of sample)
For precious sample
5 Glass bead (cell suspension microorganism)
Using abrasive vortexed bead to break cell walls
Chaotrope
Urea (up to 9 M)
Thiourea (up to 2 M)
bull Disrupt of hydrogen and hydrophobic bonds
Note Urea (if tdeggt37degC) cyanate (HN=C=O)
carbamylation (Lys Arg)
(R-NH2 R-NH-CO-NH2)
carbamylation trains
Reductants
b-mercaptoethanol
DTT(dithiothreitol)
bull Break disulfide bridge
(within or between protein)
Detergents ndash surfactants
CHAPS (up to 4)
bull Disrupt membranes
bull Break hydrophobic interactions
bull Solubilise lipids
bull Release membrane bound proteins
Note No net charge for IEF (no SDS)
Soluble in urea
Isoelectric Focusing
Measure the protein concenteration in you samples
1 Biuret
2 Lowry methods
3 Bradford methods
4 UV methods
5 Special methods
6 Other commercial methods
BCA assay (bicinchoninic acid assay Pierce)
DC protein assay (detergent compatible Bio-rad)
DCRC protein assay (detergentreducing agent compatible
Bio-rad)
Before runninng IEF you shouldhellip
Introduced by Gorg A
Ref Gorg A (1994) Westermeier (2001)
Dried gel strips can be stored at -20 to -
80 from months to years
Amersham Biosciences Bio-Rad
IPG strips (3 mm x 18 cm x 05 mm)
Narrow range
Medium range
Broad range
4 7
35 45 55 67
40 50 60
3 10
6 11
Load 400 ml sample per groove (no bubbles)
Place IPG strip gel facing down in groove
Add ~25 ml non conductive oil per groove
Peel off protective film from strip
Rehydrate overnight (~22 hrs) at room temperature
Program power supply
bull Number of gels (1-10)
bull Max voltage 5000 V
bull Vhold 125 V
bull Duration 24 hrs 00 min
bull Max current 80 mAstrip
bull Volt hours 80000 Vh
pI
+
+ - - - - - -
+ + + + + + + -
pH 4 pH 7
Cathode Anode
Place wet wicks (DH2O) under each end of strip
Set chiller temperature for 20degC (setting ~25)
Remove IPG strip from tray
Let oil drip off the strip
Place IPG strip gel facing up in equilibration tray
+ -
Add 10 ml equilibration buffer 1 per tray
+ -
+ -
+ -
+ -
(in chemical hood)
MW 4 7
+ -
10 DuracrylTM gel (22 cm x 23 cm x 1 mm)
bull TrisTricineSDS buffer
bull IPG strip
bull MW (gel worm)
bull Agarose overlay
bull Silicone spacer
bull Gasket
-
+
Conditions
bull Number of gel (1-10)
bull Max voltage 500 V
bull Max power 1600 mWgel
bull 4degC (setting 10)
bull ~19 hrs
10 DuracrylTM gel (22 cm x 23 cm x 1 mm)
MW 4 7
+ -
Trisacetate 25 mM Tris-base pH 83 (acetic acid)
200 mM Tris-base 200 mM Tricine 04 SDS
045 mm filtered
TrisTricine SDS
Bio-Rad
Digest to peptide fragment MS analysis
1 First dimension
isoelectric focusing
separation according to the pI
2 Second dimension
SDS electrophoresis (SDS-PAGE)
Separation according to the MW
Interested spot
Sensitivity Process
TimeSteps
Advantages
bullBio-safe Coomassie 10ng 25hr3 steps MS compatible easily visualised non
hazardous
bullCoomassie Blue R-250 40ng 25hr2 steps Oldest and least expensive method
bullSilver Stain Plus 1ng 15hr3 steps MS compatible high sensitivity low
background
bullBio-Rad silver stain 1ng 2hr7 steps High sensitivity detects some highly
glycosylated and other difficult to
stain proteins
bullSypro ruby 1ng 3hr2 steps MS compatible allows analysis in
flourescent imagers linear over 3
orders of magnitude
Coomassie staining
bull moderate sensitivity (36-47 ng)
bull non specific
bull not quantitative
Silver staining
bull sensitive (05-12 ng)
bull time consuming
bull non specific
bull negative stain some spots
Fluorescent dye (SYPRO ruby)
bull sensitive (1-2 ng)
bull specific quantitative
bull end point stain
Coomassie Silver
Coomassie Silver Stain Copper Stain
SDS-polyacrylamide gel
Electrotransfer to membrane
Phosphoimager for 32P and 35S labelled
1D or 2D gels
Fluoroimager for
SYPRO labelled
1D or 2D gels
Densitometer or
Photo Scanner
Normal liver
Tumor
Both
Melanie 4 (GeneBio - Windows only)
httpcaexpasyorgmelanie
ImageMaster 2D Elite (Amersham)
httpwwwimsupportcom
Phoretix 2D Advanced
httpwwwphoretixcom
PDQuest 61 (BioRad - Windows only) httpwwwproteomeworksbio-radcomhtmlpdquesthtml
Mass Spectrometry
wwwexpasych - Swiss-2DPAGE httpwwwanlgovBIOPMG - Mouse liver human breast cell lines pyrococcus Argonne Protein Mapping Group httpwwwharefieldnthamesnhsuknhliproteinindexhtml - HSC-2DPAGE Heart Science Centre Harefield Hospital httpotowustleduthcperi-gelshtm - Washington Univ Inner Ear Protein Database
httpcaexpasyorgch2d2d-indexhtml - World 2DPAGE Index of 2D gel databases
Time line ~ 4 days
bull Protein gt 250 kDa do not enter 2 SDS-PAGE properly
bull PH more than 9
bull PH less than 4
bull Hydrophobic proteins
bull Low abundant proteins
Immunoblotting Proteins from 2D gels were transferred to PVDFmembranes The membranes were blocked with 5 skimmed milk in PBS-Tween (PBS-T) for 3 h The membranes were incubated overnight at 4degC with individual sera diluted 1100 in PBS-T followed by secondary antibody (anti-human IgG conjugated with horseradish peroxidase) for 2 h with dilutions of 13000 Before and after addition of the secondary antibody membranes washed 4 times each time for 5 min in PBS-T To visualize antigenic components membranes were incubated in diaminobenzeden (DAB) and H2O2 for 15 min
List of protein spots that reacted with more than 30 of serum samples
and were identified by mass spectrometry
1Heat shock protein HSP 90-beta
2 precursor(GRP 78) 78 kDa glucose-regulated protein
3 Heat shock cognate 71 kDa protein
4 Stress-70 protein mitochondrial precursor
5 Tubulin beta-5 chain
6Tubulin alpha-6 chain and 60 kDa heat shock protein mitochondrial
precursor (Hsp60)
7 Heterogeneous nuclear ribonucleoprotein K (hnRNP K)
8 Cytokeratin 8
9 Cytokeratin 18
10 Beta-actin
11 cytokeratin 19
12 Eukaryotic initiation factor 4A-I
13 14-3-3 protein epsilon
14 14-3-3 protein zetadelta (Protein kinase Cinhibitor protein-1)
15 Translationally controlled tumor protein (TCTP)
Among these autoantigens cytokeratin 8 cytokeratin 18 Fructose-bisphosphate aldolase and enolase were reacted with more than 60 of healthy individualsrsquo serum samples These antigens were intense spots in gels
Heat Shock proteins tubulins heterogeneous nuclear ribonucleoprotein actins cytokeratins 14-3-3 protein epsilon 14-3-3 protein zetadelta eukaryotic initiation factors Rho GDP-dissociation inhibitors phosphoglycerate mutase 1 G6PD GA3PD peroxiredoxins F-acting capping protein and triosephosphate isomerase were previously reported to induce humoral arm of the immune response in cancer
Translationally controlled tumor protein and electron transfer flavoprotein alpha-subunit have not been reported to induce humoral arm of the immune response in cancer so far
In terms of biological processes the identified proteins
belong to several groups
They are mainely involved in cell motility cytoskleton
stress response apoptosis cell signaling and
metabolism
Identification of other immunogenic proteins is
underway
Appel RD et al 1993 - SWISS-2DPAGE a database of two-dimensional gel electrophoresis images Electrophoresis 14 1232-1238
Appel RD Bairoch A Sanchez JC Vargas JR Golaz O Pasquali C and Hochstrasser DF 1996 ndash Federated two-dimensional electrophoresis database a simple means of publishing two-dimensional electrophoresis data Electrophoresis 17 540-546
Bjellqvist B Ek K Righetti PG Gianazza E Gorg A Westermeier R Postel W 1982 ndash Isoelectric focusing in immobilised pH gradients principle methodology and applications JBiochemBiophysMethods 6 317-339
Brazma A et al 2001 ndash Minimum information about a microarray experiment (MIAME)-towards standards for microarray data Nat genetics 29 365-71
Hoogland C Baujard Sanchez JC Hochstrasser DF and Appel RD 1997 ndash Make2ddb a simple package to set up a two-diensional electrophoresis database for the world wide web Electrophoresis 18 2755-2758
OFarrell 1975 - High resolution two-dimensional electrophoresis of proteins JBiolChem 25 250 4007-21
Sample Preparation 2-D Electrophoresis Spot Detection amp Image Analysis
Enzymatic Digestion Peptide-Mass Fingerprinting
Peptide Sequencing via MS Database Search
Protein Identification
(I) (III)
(IV) (V) (VI)
(II)
Sample preparation buffer composition IPG strip rehydration 1st dimension IEF run IPG strip wash Equilibration buffer Equilibration buffer 2nd dimension SDS-PAGE Gel staining Immunoblot Imaging
Sample preparation
buffer composition
IPG strip rehydration
1st dimension IEF run
IPG strip wash
Equilibration buffer I
Equilibration buffer II
2nd dimension SDS-PAGE
Gel staining
Imaging
2-D Electrophoresis
Consistent protocol Limit sample degradation
bull fresh cellstissue bull protease inhibitors bull keep sample cold bull long term storage at ndash80degC
Limit sample contamination
bull gloves (keratin)
SolubilisationDenaturation buffer
bull separate proteins into individual components
bull reliable running in the IEF
Gentle lysis method 1 Osmotic lysis (cultured cells)
Suspend cells in hypoosmotic solution
2 Repeated freezing and thawing (bacteria)
Freeze using liquid nitrogen
3 Detergent lysis (yeast and fungi)
Lysis buffer (containing urea and detergent)
SDS (have to be removed before IEF)
4 Enzymatic lysis (plant bacteria fungi)
Lysomzyme (bacteria)
Cellulose and pectinase (plant)
Lyticase (yeast)
Vigorous lysis method
1 Sonication probe (cell suspension)
Avoid overheat cool on ice between burst
2 French pressure (microorganism with cell wall)
Cells are lysed by shear force
3 Mortar and pestle (solid tissue microorganism)
Grind solid tissue to fine powder with liquid nitrogen
4 Sample grinding kit (for small amount of sample)
For precious sample
5 Glass bead (cell suspension microorganism)
Using abrasive vortexed bead to break cell walls
Chaotrope
Urea (up to 9 M)
Thiourea (up to 2 M)
bull Disrupt of hydrogen and hydrophobic bonds
Note Urea (if tdeggt37degC) cyanate (HN=C=O)
carbamylation (Lys Arg)
(R-NH2 R-NH-CO-NH2)
carbamylation trains
Reductants
b-mercaptoethanol
DTT(dithiothreitol)
bull Break disulfide bridge
(within or between protein)
Detergents ndash surfactants
CHAPS (up to 4)
bull Disrupt membranes
bull Break hydrophobic interactions
bull Solubilise lipids
bull Release membrane bound proteins
Note No net charge for IEF (no SDS)
Soluble in urea
Isoelectric Focusing
Measure the protein concenteration in you samples
1 Biuret
2 Lowry methods
3 Bradford methods
4 UV methods
5 Special methods
6 Other commercial methods
BCA assay (bicinchoninic acid assay Pierce)
DC protein assay (detergent compatible Bio-rad)
DCRC protein assay (detergentreducing agent compatible
Bio-rad)
Before runninng IEF you shouldhellip
Introduced by Gorg A
Ref Gorg A (1994) Westermeier (2001)
Dried gel strips can be stored at -20 to -
80 from months to years
Amersham Biosciences Bio-Rad
IPG strips (3 mm x 18 cm x 05 mm)
Narrow range
Medium range
Broad range
4 7
35 45 55 67
40 50 60
3 10
6 11
Load 400 ml sample per groove (no bubbles)
Place IPG strip gel facing down in groove
Add ~25 ml non conductive oil per groove
Peel off protective film from strip
Rehydrate overnight (~22 hrs) at room temperature
Program power supply
bull Number of gels (1-10)
bull Max voltage 5000 V
bull Vhold 125 V
bull Duration 24 hrs 00 min
bull Max current 80 mAstrip
bull Volt hours 80000 Vh
pI
+
+ - - - - - -
+ + + + + + + -
pH 4 pH 7
Cathode Anode
Place wet wicks (DH2O) under each end of strip
Set chiller temperature for 20degC (setting ~25)
Remove IPG strip from tray
Let oil drip off the strip
Place IPG strip gel facing up in equilibration tray
+ -
Add 10 ml equilibration buffer 1 per tray
+ -
+ -
+ -
+ -
(in chemical hood)
MW 4 7
+ -
10 DuracrylTM gel (22 cm x 23 cm x 1 mm)
bull TrisTricineSDS buffer
bull IPG strip
bull MW (gel worm)
bull Agarose overlay
bull Silicone spacer
bull Gasket
-
+
Conditions
bull Number of gel (1-10)
bull Max voltage 500 V
bull Max power 1600 mWgel
bull 4degC (setting 10)
bull ~19 hrs
10 DuracrylTM gel (22 cm x 23 cm x 1 mm)
MW 4 7
+ -
Trisacetate 25 mM Tris-base pH 83 (acetic acid)
200 mM Tris-base 200 mM Tricine 04 SDS
045 mm filtered
TrisTricine SDS
Bio-Rad
Digest to peptide fragment MS analysis
1 First dimension
isoelectric focusing
separation according to the pI
2 Second dimension
SDS electrophoresis (SDS-PAGE)
Separation according to the MW
Interested spot
Sensitivity Process
TimeSteps
Advantages
bullBio-safe Coomassie 10ng 25hr3 steps MS compatible easily visualised non
hazardous
bullCoomassie Blue R-250 40ng 25hr2 steps Oldest and least expensive method
bullSilver Stain Plus 1ng 15hr3 steps MS compatible high sensitivity low
background
bullBio-Rad silver stain 1ng 2hr7 steps High sensitivity detects some highly
glycosylated and other difficult to
stain proteins
bullSypro ruby 1ng 3hr2 steps MS compatible allows analysis in
flourescent imagers linear over 3
orders of magnitude
Coomassie staining
bull moderate sensitivity (36-47 ng)
bull non specific
bull not quantitative
Silver staining
bull sensitive (05-12 ng)
bull time consuming
bull non specific
bull negative stain some spots
Fluorescent dye (SYPRO ruby)
bull sensitive (1-2 ng)
bull specific quantitative
bull end point stain
Coomassie Silver
Coomassie Silver Stain Copper Stain
SDS-polyacrylamide gel
Electrotransfer to membrane
Phosphoimager for 32P and 35S labelled
1D or 2D gels
Fluoroimager for
SYPRO labelled
1D or 2D gels
Densitometer or
Photo Scanner
Normal liver
Tumor
Both
Melanie 4 (GeneBio - Windows only)
httpcaexpasyorgmelanie
ImageMaster 2D Elite (Amersham)
httpwwwimsupportcom
Phoretix 2D Advanced
httpwwwphoretixcom
PDQuest 61 (BioRad - Windows only) httpwwwproteomeworksbio-radcomhtmlpdquesthtml
Mass Spectrometry
wwwexpasych - Swiss-2DPAGE httpwwwanlgovBIOPMG - Mouse liver human breast cell lines pyrococcus Argonne Protein Mapping Group httpwwwharefieldnthamesnhsuknhliproteinindexhtml - HSC-2DPAGE Heart Science Centre Harefield Hospital httpotowustleduthcperi-gelshtm - Washington Univ Inner Ear Protein Database
httpcaexpasyorgch2d2d-indexhtml - World 2DPAGE Index of 2D gel databases
Time line ~ 4 days
bull Protein gt 250 kDa do not enter 2 SDS-PAGE properly
bull PH more than 9
bull PH less than 4
bull Hydrophobic proteins
bull Low abundant proteins
Immunoblotting Proteins from 2D gels were transferred to PVDFmembranes The membranes were blocked with 5 skimmed milk in PBS-Tween (PBS-T) for 3 h The membranes were incubated overnight at 4degC with individual sera diluted 1100 in PBS-T followed by secondary antibody (anti-human IgG conjugated with horseradish peroxidase) for 2 h with dilutions of 13000 Before and after addition of the secondary antibody membranes washed 4 times each time for 5 min in PBS-T To visualize antigenic components membranes were incubated in diaminobenzeden (DAB) and H2O2 for 15 min
List of protein spots that reacted with more than 30 of serum samples
and were identified by mass spectrometry
1Heat shock protein HSP 90-beta
2 precursor(GRP 78) 78 kDa glucose-regulated protein
3 Heat shock cognate 71 kDa protein
4 Stress-70 protein mitochondrial precursor
5 Tubulin beta-5 chain
6Tubulin alpha-6 chain and 60 kDa heat shock protein mitochondrial
precursor (Hsp60)
7 Heterogeneous nuclear ribonucleoprotein K (hnRNP K)
8 Cytokeratin 8
9 Cytokeratin 18
10 Beta-actin
11 cytokeratin 19
12 Eukaryotic initiation factor 4A-I
13 14-3-3 protein epsilon
14 14-3-3 protein zetadelta (Protein kinase Cinhibitor protein-1)
15 Translationally controlled tumor protein (TCTP)
Among these autoantigens cytokeratin 8 cytokeratin 18 Fructose-bisphosphate aldolase and enolase were reacted with more than 60 of healthy individualsrsquo serum samples These antigens were intense spots in gels
Heat Shock proteins tubulins heterogeneous nuclear ribonucleoprotein actins cytokeratins 14-3-3 protein epsilon 14-3-3 protein zetadelta eukaryotic initiation factors Rho GDP-dissociation inhibitors phosphoglycerate mutase 1 G6PD GA3PD peroxiredoxins F-acting capping protein and triosephosphate isomerase were previously reported to induce humoral arm of the immune response in cancer
Translationally controlled tumor protein and electron transfer flavoprotein alpha-subunit have not been reported to induce humoral arm of the immune response in cancer so far
In terms of biological processes the identified proteins
belong to several groups
They are mainely involved in cell motility cytoskleton
stress response apoptosis cell signaling and
metabolism
Identification of other immunogenic proteins is
underway
Appel RD et al 1993 - SWISS-2DPAGE a database of two-dimensional gel electrophoresis images Electrophoresis 14 1232-1238
Appel RD Bairoch A Sanchez JC Vargas JR Golaz O Pasquali C and Hochstrasser DF 1996 ndash Federated two-dimensional electrophoresis database a simple means of publishing two-dimensional electrophoresis data Electrophoresis 17 540-546
Bjellqvist B Ek K Righetti PG Gianazza E Gorg A Westermeier R Postel W 1982 ndash Isoelectric focusing in immobilised pH gradients principle methodology and applications JBiochemBiophysMethods 6 317-339
Brazma A et al 2001 ndash Minimum information about a microarray experiment (MIAME)-towards standards for microarray data Nat genetics 29 365-71
Hoogland C Baujard Sanchez JC Hochstrasser DF and Appel RD 1997 ndash Make2ddb a simple package to set up a two-diensional electrophoresis database for the world wide web Electrophoresis 18 2755-2758
OFarrell 1975 - High resolution two-dimensional electrophoresis of proteins JBiolChem 25 250 4007-21
Sample preparation buffer composition IPG strip rehydration 1st dimension IEF run IPG strip wash Equilibration buffer Equilibration buffer 2nd dimension SDS-PAGE Gel staining Immunoblot Imaging
Sample preparation
buffer composition
IPG strip rehydration
1st dimension IEF run
IPG strip wash
Equilibration buffer I
Equilibration buffer II
2nd dimension SDS-PAGE
Gel staining
Imaging
2-D Electrophoresis
Consistent protocol Limit sample degradation
bull fresh cellstissue bull protease inhibitors bull keep sample cold bull long term storage at ndash80degC
Limit sample contamination
bull gloves (keratin)
SolubilisationDenaturation buffer
bull separate proteins into individual components
bull reliable running in the IEF
Gentle lysis method 1 Osmotic lysis (cultured cells)
Suspend cells in hypoosmotic solution
2 Repeated freezing and thawing (bacteria)
Freeze using liquid nitrogen
3 Detergent lysis (yeast and fungi)
Lysis buffer (containing urea and detergent)
SDS (have to be removed before IEF)
4 Enzymatic lysis (plant bacteria fungi)
Lysomzyme (bacteria)
Cellulose and pectinase (plant)
Lyticase (yeast)
Vigorous lysis method
1 Sonication probe (cell suspension)
Avoid overheat cool on ice between burst
2 French pressure (microorganism with cell wall)
Cells are lysed by shear force
3 Mortar and pestle (solid tissue microorganism)
Grind solid tissue to fine powder with liquid nitrogen
4 Sample grinding kit (for small amount of sample)
For precious sample
5 Glass bead (cell suspension microorganism)
Using abrasive vortexed bead to break cell walls
Chaotrope
Urea (up to 9 M)
Thiourea (up to 2 M)
bull Disrupt of hydrogen and hydrophobic bonds
Note Urea (if tdeggt37degC) cyanate (HN=C=O)
carbamylation (Lys Arg)
(R-NH2 R-NH-CO-NH2)
carbamylation trains
Reductants
b-mercaptoethanol
DTT(dithiothreitol)
bull Break disulfide bridge
(within or between protein)
Detergents ndash surfactants
CHAPS (up to 4)
bull Disrupt membranes
bull Break hydrophobic interactions
bull Solubilise lipids
bull Release membrane bound proteins
Note No net charge for IEF (no SDS)
Soluble in urea
Isoelectric Focusing
Measure the protein concenteration in you samples
1 Biuret
2 Lowry methods
3 Bradford methods
4 UV methods
5 Special methods
6 Other commercial methods
BCA assay (bicinchoninic acid assay Pierce)
DC protein assay (detergent compatible Bio-rad)
DCRC protein assay (detergentreducing agent compatible
Bio-rad)
Before runninng IEF you shouldhellip
Introduced by Gorg A
Ref Gorg A (1994) Westermeier (2001)
Dried gel strips can be stored at -20 to -
80 from months to years
Amersham Biosciences Bio-Rad
IPG strips (3 mm x 18 cm x 05 mm)
Narrow range
Medium range
Broad range
4 7
35 45 55 67
40 50 60
3 10
6 11
Load 400 ml sample per groove (no bubbles)
Place IPG strip gel facing down in groove
Add ~25 ml non conductive oil per groove
Peel off protective film from strip
Rehydrate overnight (~22 hrs) at room temperature
Program power supply
bull Number of gels (1-10)
bull Max voltage 5000 V
bull Vhold 125 V
bull Duration 24 hrs 00 min
bull Max current 80 mAstrip
bull Volt hours 80000 Vh
pI
+
+ - - - - - -
+ + + + + + + -
pH 4 pH 7
Cathode Anode
Place wet wicks (DH2O) under each end of strip
Set chiller temperature for 20degC (setting ~25)
Remove IPG strip from tray
Let oil drip off the strip
Place IPG strip gel facing up in equilibration tray
+ -
Add 10 ml equilibration buffer 1 per tray
+ -
+ -
+ -
+ -
(in chemical hood)
MW 4 7
+ -
10 DuracrylTM gel (22 cm x 23 cm x 1 mm)
bull TrisTricineSDS buffer
bull IPG strip
bull MW (gel worm)
bull Agarose overlay
bull Silicone spacer
bull Gasket
-
+
Conditions
bull Number of gel (1-10)
bull Max voltage 500 V
bull Max power 1600 mWgel
bull 4degC (setting 10)
bull ~19 hrs
10 DuracrylTM gel (22 cm x 23 cm x 1 mm)
MW 4 7
+ -
Trisacetate 25 mM Tris-base pH 83 (acetic acid)
200 mM Tris-base 200 mM Tricine 04 SDS
045 mm filtered
TrisTricine SDS
Bio-Rad
Digest to peptide fragment MS analysis
1 First dimension
isoelectric focusing
separation according to the pI
2 Second dimension
SDS electrophoresis (SDS-PAGE)
Separation according to the MW
Interested spot
Sensitivity Process
TimeSteps
Advantages
bullBio-safe Coomassie 10ng 25hr3 steps MS compatible easily visualised non
hazardous
bullCoomassie Blue R-250 40ng 25hr2 steps Oldest and least expensive method
bullSilver Stain Plus 1ng 15hr3 steps MS compatible high sensitivity low
background
bullBio-Rad silver stain 1ng 2hr7 steps High sensitivity detects some highly
glycosylated and other difficult to
stain proteins
bullSypro ruby 1ng 3hr2 steps MS compatible allows analysis in
flourescent imagers linear over 3
orders of magnitude
Coomassie staining
bull moderate sensitivity (36-47 ng)
bull non specific
bull not quantitative
Silver staining
bull sensitive (05-12 ng)
bull time consuming
bull non specific
bull negative stain some spots
Fluorescent dye (SYPRO ruby)
bull sensitive (1-2 ng)
bull specific quantitative
bull end point stain
Coomassie Silver
Coomassie Silver Stain Copper Stain
SDS-polyacrylamide gel
Electrotransfer to membrane
Phosphoimager for 32P and 35S labelled
1D or 2D gels
Fluoroimager for
SYPRO labelled
1D or 2D gels
Densitometer or
Photo Scanner
Normal liver
Tumor
Both
Melanie 4 (GeneBio - Windows only)
httpcaexpasyorgmelanie
ImageMaster 2D Elite (Amersham)
httpwwwimsupportcom
Phoretix 2D Advanced
httpwwwphoretixcom
PDQuest 61 (BioRad - Windows only) httpwwwproteomeworksbio-radcomhtmlpdquesthtml
Mass Spectrometry
wwwexpasych - Swiss-2DPAGE httpwwwanlgovBIOPMG - Mouse liver human breast cell lines pyrococcus Argonne Protein Mapping Group httpwwwharefieldnthamesnhsuknhliproteinindexhtml - HSC-2DPAGE Heart Science Centre Harefield Hospital httpotowustleduthcperi-gelshtm - Washington Univ Inner Ear Protein Database
httpcaexpasyorgch2d2d-indexhtml - World 2DPAGE Index of 2D gel databases
Time line ~ 4 days
bull Protein gt 250 kDa do not enter 2 SDS-PAGE properly
bull PH more than 9
bull PH less than 4
bull Hydrophobic proteins
bull Low abundant proteins
Immunoblotting Proteins from 2D gels were transferred to PVDFmembranes The membranes were blocked with 5 skimmed milk in PBS-Tween (PBS-T) for 3 h The membranes were incubated overnight at 4degC with individual sera diluted 1100 in PBS-T followed by secondary antibody (anti-human IgG conjugated with horseradish peroxidase) for 2 h with dilutions of 13000 Before and after addition of the secondary antibody membranes washed 4 times each time for 5 min in PBS-T To visualize antigenic components membranes were incubated in diaminobenzeden (DAB) and H2O2 for 15 min
List of protein spots that reacted with more than 30 of serum samples
and were identified by mass spectrometry
1Heat shock protein HSP 90-beta
2 precursor(GRP 78) 78 kDa glucose-regulated protein
3 Heat shock cognate 71 kDa protein
4 Stress-70 protein mitochondrial precursor
5 Tubulin beta-5 chain
6Tubulin alpha-6 chain and 60 kDa heat shock protein mitochondrial
precursor (Hsp60)
7 Heterogeneous nuclear ribonucleoprotein K (hnRNP K)
8 Cytokeratin 8
9 Cytokeratin 18
10 Beta-actin
11 cytokeratin 19
12 Eukaryotic initiation factor 4A-I
13 14-3-3 protein epsilon
14 14-3-3 protein zetadelta (Protein kinase Cinhibitor protein-1)
15 Translationally controlled tumor protein (TCTP)
Among these autoantigens cytokeratin 8 cytokeratin 18 Fructose-bisphosphate aldolase and enolase were reacted with more than 60 of healthy individualsrsquo serum samples These antigens were intense spots in gels
Heat Shock proteins tubulins heterogeneous nuclear ribonucleoprotein actins cytokeratins 14-3-3 protein epsilon 14-3-3 protein zetadelta eukaryotic initiation factors Rho GDP-dissociation inhibitors phosphoglycerate mutase 1 G6PD GA3PD peroxiredoxins F-acting capping protein and triosephosphate isomerase were previously reported to induce humoral arm of the immune response in cancer
Translationally controlled tumor protein and electron transfer flavoprotein alpha-subunit have not been reported to induce humoral arm of the immune response in cancer so far
In terms of biological processes the identified proteins
belong to several groups
They are mainely involved in cell motility cytoskleton
stress response apoptosis cell signaling and
metabolism
Identification of other immunogenic proteins is
underway
Appel RD et al 1993 - SWISS-2DPAGE a database of two-dimensional gel electrophoresis images Electrophoresis 14 1232-1238
Appel RD Bairoch A Sanchez JC Vargas JR Golaz O Pasquali C and Hochstrasser DF 1996 ndash Federated two-dimensional electrophoresis database a simple means of publishing two-dimensional electrophoresis data Electrophoresis 17 540-546
Bjellqvist B Ek K Righetti PG Gianazza E Gorg A Westermeier R Postel W 1982 ndash Isoelectric focusing in immobilised pH gradients principle methodology and applications JBiochemBiophysMethods 6 317-339
Brazma A et al 2001 ndash Minimum information about a microarray experiment (MIAME)-towards standards for microarray data Nat genetics 29 365-71
Hoogland C Baujard Sanchez JC Hochstrasser DF and Appel RD 1997 ndash Make2ddb a simple package to set up a two-diensional electrophoresis database for the world wide web Electrophoresis 18 2755-2758
OFarrell 1975 - High resolution two-dimensional electrophoresis of proteins JBiolChem 25 250 4007-21
Consistent protocol Limit sample degradation
bull fresh cellstissue bull protease inhibitors bull keep sample cold bull long term storage at ndash80degC
Limit sample contamination
bull gloves (keratin)
SolubilisationDenaturation buffer
bull separate proteins into individual components
bull reliable running in the IEF
Gentle lysis method 1 Osmotic lysis (cultured cells)
Suspend cells in hypoosmotic solution
2 Repeated freezing and thawing (bacteria)
Freeze using liquid nitrogen
3 Detergent lysis (yeast and fungi)
Lysis buffer (containing urea and detergent)
SDS (have to be removed before IEF)
4 Enzymatic lysis (plant bacteria fungi)
Lysomzyme (bacteria)
Cellulose and pectinase (plant)
Lyticase (yeast)
Vigorous lysis method
1 Sonication probe (cell suspension)
Avoid overheat cool on ice between burst
2 French pressure (microorganism with cell wall)
Cells are lysed by shear force
3 Mortar and pestle (solid tissue microorganism)
Grind solid tissue to fine powder with liquid nitrogen
4 Sample grinding kit (for small amount of sample)
For precious sample
5 Glass bead (cell suspension microorganism)
Using abrasive vortexed bead to break cell walls
Chaotrope
Urea (up to 9 M)
Thiourea (up to 2 M)
bull Disrupt of hydrogen and hydrophobic bonds
Note Urea (if tdeggt37degC) cyanate (HN=C=O)
carbamylation (Lys Arg)
(R-NH2 R-NH-CO-NH2)
carbamylation trains
Reductants
b-mercaptoethanol
DTT(dithiothreitol)
bull Break disulfide bridge
(within or between protein)
Detergents ndash surfactants
CHAPS (up to 4)
bull Disrupt membranes
bull Break hydrophobic interactions
bull Solubilise lipids
bull Release membrane bound proteins
Note No net charge for IEF (no SDS)
Soluble in urea
Isoelectric Focusing
Measure the protein concenteration in you samples
1 Biuret
2 Lowry methods
3 Bradford methods
4 UV methods
5 Special methods
6 Other commercial methods
BCA assay (bicinchoninic acid assay Pierce)
DC protein assay (detergent compatible Bio-rad)
DCRC protein assay (detergentreducing agent compatible
Bio-rad)
Before runninng IEF you shouldhellip
Introduced by Gorg A
Ref Gorg A (1994) Westermeier (2001)
Dried gel strips can be stored at -20 to -
80 from months to years
Amersham Biosciences Bio-Rad
IPG strips (3 mm x 18 cm x 05 mm)
Narrow range
Medium range
Broad range
4 7
35 45 55 67
40 50 60
3 10
6 11
Load 400 ml sample per groove (no bubbles)
Place IPG strip gel facing down in groove
Add ~25 ml non conductive oil per groove
Peel off protective film from strip
Rehydrate overnight (~22 hrs) at room temperature
Program power supply
bull Number of gels (1-10)
bull Max voltage 5000 V
bull Vhold 125 V
bull Duration 24 hrs 00 min
bull Max current 80 mAstrip
bull Volt hours 80000 Vh
pI
+
+ - - - - - -
+ + + + + + + -
pH 4 pH 7
Cathode Anode
Place wet wicks (DH2O) under each end of strip
Set chiller temperature for 20degC (setting ~25)
Remove IPG strip from tray
Let oil drip off the strip
Place IPG strip gel facing up in equilibration tray
+ -
Add 10 ml equilibration buffer 1 per tray
+ -
+ -
+ -
+ -
(in chemical hood)
MW 4 7
+ -
10 DuracrylTM gel (22 cm x 23 cm x 1 mm)
bull TrisTricineSDS buffer
bull IPG strip
bull MW (gel worm)
bull Agarose overlay
bull Silicone spacer
bull Gasket
-
+
Conditions
bull Number of gel (1-10)
bull Max voltage 500 V
bull Max power 1600 mWgel
bull 4degC (setting 10)
bull ~19 hrs
10 DuracrylTM gel (22 cm x 23 cm x 1 mm)
MW 4 7
+ -
Trisacetate 25 mM Tris-base pH 83 (acetic acid)
200 mM Tris-base 200 mM Tricine 04 SDS
045 mm filtered
TrisTricine SDS
Bio-Rad
Digest to peptide fragment MS analysis
1 First dimension
isoelectric focusing
separation according to the pI
2 Second dimension
SDS electrophoresis (SDS-PAGE)
Separation according to the MW
Interested spot
Sensitivity Process
TimeSteps
Advantages
bullBio-safe Coomassie 10ng 25hr3 steps MS compatible easily visualised non
hazardous
bullCoomassie Blue R-250 40ng 25hr2 steps Oldest and least expensive method
bullSilver Stain Plus 1ng 15hr3 steps MS compatible high sensitivity low
background
bullBio-Rad silver stain 1ng 2hr7 steps High sensitivity detects some highly
glycosylated and other difficult to
stain proteins
bullSypro ruby 1ng 3hr2 steps MS compatible allows analysis in
flourescent imagers linear over 3
orders of magnitude
Coomassie staining
bull moderate sensitivity (36-47 ng)
bull non specific
bull not quantitative
Silver staining
bull sensitive (05-12 ng)
bull time consuming
bull non specific
bull negative stain some spots
Fluorescent dye (SYPRO ruby)
bull sensitive (1-2 ng)
bull specific quantitative
bull end point stain
Coomassie Silver
Coomassie Silver Stain Copper Stain
SDS-polyacrylamide gel
Electrotransfer to membrane
Phosphoimager for 32P and 35S labelled
1D or 2D gels
Fluoroimager for
SYPRO labelled
1D or 2D gels
Densitometer or
Photo Scanner
Normal liver
Tumor
Both
Melanie 4 (GeneBio - Windows only)
httpcaexpasyorgmelanie
ImageMaster 2D Elite (Amersham)
httpwwwimsupportcom
Phoretix 2D Advanced
httpwwwphoretixcom
PDQuest 61 (BioRad - Windows only) httpwwwproteomeworksbio-radcomhtmlpdquesthtml
Mass Spectrometry
wwwexpasych - Swiss-2DPAGE httpwwwanlgovBIOPMG - Mouse liver human breast cell lines pyrococcus Argonne Protein Mapping Group httpwwwharefieldnthamesnhsuknhliproteinindexhtml - HSC-2DPAGE Heart Science Centre Harefield Hospital httpotowustleduthcperi-gelshtm - Washington Univ Inner Ear Protein Database
httpcaexpasyorgch2d2d-indexhtml - World 2DPAGE Index of 2D gel databases
Time line ~ 4 days
bull Protein gt 250 kDa do not enter 2 SDS-PAGE properly
bull PH more than 9
bull PH less than 4
bull Hydrophobic proteins
bull Low abundant proteins
Immunoblotting Proteins from 2D gels were transferred to PVDFmembranes The membranes were blocked with 5 skimmed milk in PBS-Tween (PBS-T) for 3 h The membranes were incubated overnight at 4degC with individual sera diluted 1100 in PBS-T followed by secondary antibody (anti-human IgG conjugated with horseradish peroxidase) for 2 h with dilutions of 13000 Before and after addition of the secondary antibody membranes washed 4 times each time for 5 min in PBS-T To visualize antigenic components membranes were incubated in diaminobenzeden (DAB) and H2O2 for 15 min
List of protein spots that reacted with more than 30 of serum samples
and were identified by mass spectrometry
1Heat shock protein HSP 90-beta
2 precursor(GRP 78) 78 kDa glucose-regulated protein
3 Heat shock cognate 71 kDa protein
4 Stress-70 protein mitochondrial precursor
5 Tubulin beta-5 chain
6Tubulin alpha-6 chain and 60 kDa heat shock protein mitochondrial
precursor (Hsp60)
7 Heterogeneous nuclear ribonucleoprotein K (hnRNP K)
8 Cytokeratin 8
9 Cytokeratin 18
10 Beta-actin
11 cytokeratin 19
12 Eukaryotic initiation factor 4A-I
13 14-3-3 protein epsilon
14 14-3-3 protein zetadelta (Protein kinase Cinhibitor protein-1)
15 Translationally controlled tumor protein (TCTP)
Among these autoantigens cytokeratin 8 cytokeratin 18 Fructose-bisphosphate aldolase and enolase were reacted with more than 60 of healthy individualsrsquo serum samples These antigens were intense spots in gels
Heat Shock proteins tubulins heterogeneous nuclear ribonucleoprotein actins cytokeratins 14-3-3 protein epsilon 14-3-3 protein zetadelta eukaryotic initiation factors Rho GDP-dissociation inhibitors phosphoglycerate mutase 1 G6PD GA3PD peroxiredoxins F-acting capping protein and triosephosphate isomerase were previously reported to induce humoral arm of the immune response in cancer
Translationally controlled tumor protein and electron transfer flavoprotein alpha-subunit have not been reported to induce humoral arm of the immune response in cancer so far
In terms of biological processes the identified proteins
belong to several groups
They are mainely involved in cell motility cytoskleton
stress response apoptosis cell signaling and
metabolism
Identification of other immunogenic proteins is
underway
Appel RD et al 1993 - SWISS-2DPAGE a database of two-dimensional gel electrophoresis images Electrophoresis 14 1232-1238
Appel RD Bairoch A Sanchez JC Vargas JR Golaz O Pasquali C and Hochstrasser DF 1996 ndash Federated two-dimensional electrophoresis database a simple means of publishing two-dimensional electrophoresis data Electrophoresis 17 540-546
Bjellqvist B Ek K Righetti PG Gianazza E Gorg A Westermeier R Postel W 1982 ndash Isoelectric focusing in immobilised pH gradients principle methodology and applications JBiochemBiophysMethods 6 317-339
Brazma A et al 2001 ndash Minimum information about a microarray experiment (MIAME)-towards standards for microarray data Nat genetics 29 365-71
Hoogland C Baujard Sanchez JC Hochstrasser DF and Appel RD 1997 ndash Make2ddb a simple package to set up a two-diensional electrophoresis database for the world wide web Electrophoresis 18 2755-2758
OFarrell 1975 - High resolution two-dimensional electrophoresis of proteins JBiolChem 25 250 4007-21
SolubilisationDenaturation buffer
bull separate proteins into individual components
bull reliable running in the IEF
Gentle lysis method 1 Osmotic lysis (cultured cells)
Suspend cells in hypoosmotic solution
2 Repeated freezing and thawing (bacteria)
Freeze using liquid nitrogen
3 Detergent lysis (yeast and fungi)
Lysis buffer (containing urea and detergent)
SDS (have to be removed before IEF)
4 Enzymatic lysis (plant bacteria fungi)
Lysomzyme (bacteria)
Cellulose and pectinase (plant)
Lyticase (yeast)
Vigorous lysis method
1 Sonication probe (cell suspension)
Avoid overheat cool on ice between burst
2 French pressure (microorganism with cell wall)
Cells are lysed by shear force
3 Mortar and pestle (solid tissue microorganism)
Grind solid tissue to fine powder with liquid nitrogen
4 Sample grinding kit (for small amount of sample)
For precious sample
5 Glass bead (cell suspension microorganism)
Using abrasive vortexed bead to break cell walls
Chaotrope
Urea (up to 9 M)
Thiourea (up to 2 M)
bull Disrupt of hydrogen and hydrophobic bonds
Note Urea (if tdeggt37degC) cyanate (HN=C=O)
carbamylation (Lys Arg)
(R-NH2 R-NH-CO-NH2)
carbamylation trains
Reductants
b-mercaptoethanol
DTT(dithiothreitol)
bull Break disulfide bridge
(within or between protein)
Detergents ndash surfactants
CHAPS (up to 4)
bull Disrupt membranes
bull Break hydrophobic interactions
bull Solubilise lipids
bull Release membrane bound proteins
Note No net charge for IEF (no SDS)
Soluble in urea
Isoelectric Focusing
Measure the protein concenteration in you samples
1 Biuret
2 Lowry methods
3 Bradford methods
4 UV methods
5 Special methods
6 Other commercial methods
BCA assay (bicinchoninic acid assay Pierce)
DC protein assay (detergent compatible Bio-rad)
DCRC protein assay (detergentreducing agent compatible
Bio-rad)
Before runninng IEF you shouldhellip
Introduced by Gorg A
Ref Gorg A (1994) Westermeier (2001)
Dried gel strips can be stored at -20 to -
80 from months to years
Amersham Biosciences Bio-Rad
IPG strips (3 mm x 18 cm x 05 mm)
Narrow range
Medium range
Broad range
4 7
35 45 55 67
40 50 60
3 10
6 11
Load 400 ml sample per groove (no bubbles)
Place IPG strip gel facing down in groove
Add ~25 ml non conductive oil per groove
Peel off protective film from strip
Rehydrate overnight (~22 hrs) at room temperature
Program power supply
bull Number of gels (1-10)
bull Max voltage 5000 V
bull Vhold 125 V
bull Duration 24 hrs 00 min
bull Max current 80 mAstrip
bull Volt hours 80000 Vh
pI
+
+ - - - - - -
+ + + + + + + -
pH 4 pH 7
Cathode Anode
Place wet wicks (DH2O) under each end of strip
Set chiller temperature for 20degC (setting ~25)
Remove IPG strip from tray
Let oil drip off the strip
Place IPG strip gel facing up in equilibration tray
+ -
Add 10 ml equilibration buffer 1 per tray
+ -
+ -
+ -
+ -
(in chemical hood)
MW 4 7
+ -
10 DuracrylTM gel (22 cm x 23 cm x 1 mm)
bull TrisTricineSDS buffer
bull IPG strip
bull MW (gel worm)
bull Agarose overlay
bull Silicone spacer
bull Gasket
-
+
Conditions
bull Number of gel (1-10)
bull Max voltage 500 V
bull Max power 1600 mWgel
bull 4degC (setting 10)
bull ~19 hrs
10 DuracrylTM gel (22 cm x 23 cm x 1 mm)
MW 4 7
+ -
Trisacetate 25 mM Tris-base pH 83 (acetic acid)
200 mM Tris-base 200 mM Tricine 04 SDS
045 mm filtered
TrisTricine SDS
Bio-Rad
Digest to peptide fragment MS analysis
1 First dimension
isoelectric focusing
separation according to the pI
2 Second dimension
SDS electrophoresis (SDS-PAGE)
Separation according to the MW
Interested spot
Sensitivity Process
TimeSteps
Advantages
bullBio-safe Coomassie 10ng 25hr3 steps MS compatible easily visualised non
hazardous
bullCoomassie Blue R-250 40ng 25hr2 steps Oldest and least expensive method
bullSilver Stain Plus 1ng 15hr3 steps MS compatible high sensitivity low
background
bullBio-Rad silver stain 1ng 2hr7 steps High sensitivity detects some highly
glycosylated and other difficult to
stain proteins
bullSypro ruby 1ng 3hr2 steps MS compatible allows analysis in
flourescent imagers linear over 3
orders of magnitude
Coomassie staining
bull moderate sensitivity (36-47 ng)
bull non specific
bull not quantitative
Silver staining
bull sensitive (05-12 ng)
bull time consuming
bull non specific
bull negative stain some spots
Fluorescent dye (SYPRO ruby)
bull sensitive (1-2 ng)
bull specific quantitative
bull end point stain
Coomassie Silver
Coomassie Silver Stain Copper Stain
SDS-polyacrylamide gel
Electrotransfer to membrane
Phosphoimager for 32P and 35S labelled
1D or 2D gels
Fluoroimager for
SYPRO labelled
1D or 2D gels
Densitometer or
Photo Scanner
Normal liver
Tumor
Both
Melanie 4 (GeneBio - Windows only)
httpcaexpasyorgmelanie
ImageMaster 2D Elite (Amersham)
httpwwwimsupportcom
Phoretix 2D Advanced
httpwwwphoretixcom
PDQuest 61 (BioRad - Windows only) httpwwwproteomeworksbio-radcomhtmlpdquesthtml
Mass Spectrometry
wwwexpasych - Swiss-2DPAGE httpwwwanlgovBIOPMG - Mouse liver human breast cell lines pyrococcus Argonne Protein Mapping Group httpwwwharefieldnthamesnhsuknhliproteinindexhtml - HSC-2DPAGE Heart Science Centre Harefield Hospital httpotowustleduthcperi-gelshtm - Washington Univ Inner Ear Protein Database
httpcaexpasyorgch2d2d-indexhtml - World 2DPAGE Index of 2D gel databases
Time line ~ 4 days
bull Protein gt 250 kDa do not enter 2 SDS-PAGE properly
bull PH more than 9
bull PH less than 4
bull Hydrophobic proteins
bull Low abundant proteins
Immunoblotting Proteins from 2D gels were transferred to PVDFmembranes The membranes were blocked with 5 skimmed milk in PBS-Tween (PBS-T) for 3 h The membranes were incubated overnight at 4degC with individual sera diluted 1100 in PBS-T followed by secondary antibody (anti-human IgG conjugated with horseradish peroxidase) for 2 h with dilutions of 13000 Before and after addition of the secondary antibody membranes washed 4 times each time for 5 min in PBS-T To visualize antigenic components membranes were incubated in diaminobenzeden (DAB) and H2O2 for 15 min
List of protein spots that reacted with more than 30 of serum samples
and were identified by mass spectrometry
1Heat shock protein HSP 90-beta
2 precursor(GRP 78) 78 kDa glucose-regulated protein
3 Heat shock cognate 71 kDa protein
4 Stress-70 protein mitochondrial precursor
5 Tubulin beta-5 chain
6Tubulin alpha-6 chain and 60 kDa heat shock protein mitochondrial
precursor (Hsp60)
7 Heterogeneous nuclear ribonucleoprotein K (hnRNP K)
8 Cytokeratin 8
9 Cytokeratin 18
10 Beta-actin
11 cytokeratin 19
12 Eukaryotic initiation factor 4A-I
13 14-3-3 protein epsilon
14 14-3-3 protein zetadelta (Protein kinase Cinhibitor protein-1)
15 Translationally controlled tumor protein (TCTP)
Among these autoantigens cytokeratin 8 cytokeratin 18 Fructose-bisphosphate aldolase and enolase were reacted with more than 60 of healthy individualsrsquo serum samples These antigens were intense spots in gels
Heat Shock proteins tubulins heterogeneous nuclear ribonucleoprotein actins cytokeratins 14-3-3 protein epsilon 14-3-3 protein zetadelta eukaryotic initiation factors Rho GDP-dissociation inhibitors phosphoglycerate mutase 1 G6PD GA3PD peroxiredoxins F-acting capping protein and triosephosphate isomerase were previously reported to induce humoral arm of the immune response in cancer
Translationally controlled tumor protein and electron transfer flavoprotein alpha-subunit have not been reported to induce humoral arm of the immune response in cancer so far
In terms of biological processes the identified proteins
belong to several groups
They are mainely involved in cell motility cytoskleton
stress response apoptosis cell signaling and
metabolism
Identification of other immunogenic proteins is
underway
Appel RD et al 1993 - SWISS-2DPAGE a database of two-dimensional gel electrophoresis images Electrophoresis 14 1232-1238
Appel RD Bairoch A Sanchez JC Vargas JR Golaz O Pasquali C and Hochstrasser DF 1996 ndash Federated two-dimensional electrophoresis database a simple means of publishing two-dimensional electrophoresis data Electrophoresis 17 540-546
Bjellqvist B Ek K Righetti PG Gianazza E Gorg A Westermeier R Postel W 1982 ndash Isoelectric focusing in immobilised pH gradients principle methodology and applications JBiochemBiophysMethods 6 317-339
Brazma A et al 2001 ndash Minimum information about a microarray experiment (MIAME)-towards standards for microarray data Nat genetics 29 365-71
Hoogland C Baujard Sanchez JC Hochstrasser DF and Appel RD 1997 ndash Make2ddb a simple package to set up a two-diensional electrophoresis database for the world wide web Electrophoresis 18 2755-2758
OFarrell 1975 - High resolution two-dimensional electrophoresis of proteins JBiolChem 25 250 4007-21
Gentle lysis method 1 Osmotic lysis (cultured cells)
Suspend cells in hypoosmotic solution
2 Repeated freezing and thawing (bacteria)
Freeze using liquid nitrogen
3 Detergent lysis (yeast and fungi)
Lysis buffer (containing urea and detergent)
SDS (have to be removed before IEF)
4 Enzymatic lysis (plant bacteria fungi)
Lysomzyme (bacteria)
Cellulose and pectinase (plant)
Lyticase (yeast)
Vigorous lysis method
1 Sonication probe (cell suspension)
Avoid overheat cool on ice between burst
2 French pressure (microorganism with cell wall)
Cells are lysed by shear force
3 Mortar and pestle (solid tissue microorganism)
Grind solid tissue to fine powder with liquid nitrogen
4 Sample grinding kit (for small amount of sample)
For precious sample
5 Glass bead (cell suspension microorganism)
Using abrasive vortexed bead to break cell walls
Chaotrope
Urea (up to 9 M)
Thiourea (up to 2 M)
bull Disrupt of hydrogen and hydrophobic bonds
Note Urea (if tdeggt37degC) cyanate (HN=C=O)
carbamylation (Lys Arg)
(R-NH2 R-NH-CO-NH2)
carbamylation trains
Reductants
b-mercaptoethanol
DTT(dithiothreitol)
bull Break disulfide bridge
(within or between protein)
Detergents ndash surfactants
CHAPS (up to 4)
bull Disrupt membranes
bull Break hydrophobic interactions
bull Solubilise lipids
bull Release membrane bound proteins
Note No net charge for IEF (no SDS)
Soluble in urea
Isoelectric Focusing
Measure the protein concenteration in you samples
1 Biuret
2 Lowry methods
3 Bradford methods
4 UV methods
5 Special methods
6 Other commercial methods
BCA assay (bicinchoninic acid assay Pierce)
DC protein assay (detergent compatible Bio-rad)
DCRC protein assay (detergentreducing agent compatible
Bio-rad)
Before runninng IEF you shouldhellip
Introduced by Gorg A
Ref Gorg A (1994) Westermeier (2001)
Dried gel strips can be stored at -20 to -
80 from months to years
Amersham Biosciences Bio-Rad
IPG strips (3 mm x 18 cm x 05 mm)
Narrow range
Medium range
Broad range
4 7
35 45 55 67
40 50 60
3 10
6 11
Load 400 ml sample per groove (no bubbles)
Place IPG strip gel facing down in groove
Add ~25 ml non conductive oil per groove
Peel off protective film from strip
Rehydrate overnight (~22 hrs) at room temperature
Program power supply
bull Number of gels (1-10)
bull Max voltage 5000 V
bull Vhold 125 V
bull Duration 24 hrs 00 min
bull Max current 80 mAstrip
bull Volt hours 80000 Vh
pI
+
+ - - - - - -
+ + + + + + + -
pH 4 pH 7
Cathode Anode
Place wet wicks (DH2O) under each end of strip
Set chiller temperature for 20degC (setting ~25)
Remove IPG strip from tray
Let oil drip off the strip
Place IPG strip gel facing up in equilibration tray
+ -
Add 10 ml equilibration buffer 1 per tray
+ -
+ -
+ -
+ -
(in chemical hood)
MW 4 7
+ -
10 DuracrylTM gel (22 cm x 23 cm x 1 mm)
bull TrisTricineSDS buffer
bull IPG strip
bull MW (gel worm)
bull Agarose overlay
bull Silicone spacer
bull Gasket
-
+
Conditions
bull Number of gel (1-10)
bull Max voltage 500 V
bull Max power 1600 mWgel
bull 4degC (setting 10)
bull ~19 hrs
10 DuracrylTM gel (22 cm x 23 cm x 1 mm)
MW 4 7
+ -
Trisacetate 25 mM Tris-base pH 83 (acetic acid)
200 mM Tris-base 200 mM Tricine 04 SDS
045 mm filtered
TrisTricine SDS
Bio-Rad
Digest to peptide fragment MS analysis
1 First dimension
isoelectric focusing
separation according to the pI
2 Second dimension
SDS electrophoresis (SDS-PAGE)
Separation according to the MW
Interested spot
Sensitivity Process
TimeSteps
Advantages
bullBio-safe Coomassie 10ng 25hr3 steps MS compatible easily visualised non
hazardous
bullCoomassie Blue R-250 40ng 25hr2 steps Oldest and least expensive method
bullSilver Stain Plus 1ng 15hr3 steps MS compatible high sensitivity low
background
bullBio-Rad silver stain 1ng 2hr7 steps High sensitivity detects some highly
glycosylated and other difficult to
stain proteins
bullSypro ruby 1ng 3hr2 steps MS compatible allows analysis in
flourescent imagers linear over 3
orders of magnitude
Coomassie staining
bull moderate sensitivity (36-47 ng)
bull non specific
bull not quantitative
Silver staining
bull sensitive (05-12 ng)
bull time consuming
bull non specific
bull negative stain some spots
Fluorescent dye (SYPRO ruby)
bull sensitive (1-2 ng)
bull specific quantitative
bull end point stain
Coomassie Silver
Coomassie Silver Stain Copper Stain
SDS-polyacrylamide gel
Electrotransfer to membrane
Phosphoimager for 32P and 35S labelled
1D or 2D gels
Fluoroimager for
SYPRO labelled
1D or 2D gels
Densitometer or
Photo Scanner
Normal liver
Tumor
Both
Melanie 4 (GeneBio - Windows only)
httpcaexpasyorgmelanie
ImageMaster 2D Elite (Amersham)
httpwwwimsupportcom
Phoretix 2D Advanced
httpwwwphoretixcom
PDQuest 61 (BioRad - Windows only) httpwwwproteomeworksbio-radcomhtmlpdquesthtml
Mass Spectrometry
wwwexpasych - Swiss-2DPAGE httpwwwanlgovBIOPMG - Mouse liver human breast cell lines pyrococcus Argonne Protein Mapping Group httpwwwharefieldnthamesnhsuknhliproteinindexhtml - HSC-2DPAGE Heart Science Centre Harefield Hospital httpotowustleduthcperi-gelshtm - Washington Univ Inner Ear Protein Database
httpcaexpasyorgch2d2d-indexhtml - World 2DPAGE Index of 2D gel databases
Time line ~ 4 days
bull Protein gt 250 kDa do not enter 2 SDS-PAGE properly
bull PH more than 9
bull PH less than 4
bull Hydrophobic proteins
bull Low abundant proteins
Immunoblotting Proteins from 2D gels were transferred to PVDFmembranes The membranes were blocked with 5 skimmed milk in PBS-Tween (PBS-T) for 3 h The membranes were incubated overnight at 4degC with individual sera diluted 1100 in PBS-T followed by secondary antibody (anti-human IgG conjugated with horseradish peroxidase) for 2 h with dilutions of 13000 Before and after addition of the secondary antibody membranes washed 4 times each time for 5 min in PBS-T To visualize antigenic components membranes were incubated in diaminobenzeden (DAB) and H2O2 for 15 min
List of protein spots that reacted with more than 30 of serum samples
and were identified by mass spectrometry
1Heat shock protein HSP 90-beta
2 precursor(GRP 78) 78 kDa glucose-regulated protein
3 Heat shock cognate 71 kDa protein
4 Stress-70 protein mitochondrial precursor
5 Tubulin beta-5 chain
6Tubulin alpha-6 chain and 60 kDa heat shock protein mitochondrial
precursor (Hsp60)
7 Heterogeneous nuclear ribonucleoprotein K (hnRNP K)
8 Cytokeratin 8
9 Cytokeratin 18
10 Beta-actin
11 cytokeratin 19
12 Eukaryotic initiation factor 4A-I
13 14-3-3 protein epsilon
14 14-3-3 protein zetadelta (Protein kinase Cinhibitor protein-1)
15 Translationally controlled tumor protein (TCTP)
Among these autoantigens cytokeratin 8 cytokeratin 18 Fructose-bisphosphate aldolase and enolase were reacted with more than 60 of healthy individualsrsquo serum samples These antigens were intense spots in gels
Heat Shock proteins tubulins heterogeneous nuclear ribonucleoprotein actins cytokeratins 14-3-3 protein epsilon 14-3-3 protein zetadelta eukaryotic initiation factors Rho GDP-dissociation inhibitors phosphoglycerate mutase 1 G6PD GA3PD peroxiredoxins F-acting capping protein and triosephosphate isomerase were previously reported to induce humoral arm of the immune response in cancer
Translationally controlled tumor protein and electron transfer flavoprotein alpha-subunit have not been reported to induce humoral arm of the immune response in cancer so far
In terms of biological processes the identified proteins
belong to several groups
They are mainely involved in cell motility cytoskleton
stress response apoptosis cell signaling and
metabolism
Identification of other immunogenic proteins is
underway
Appel RD et al 1993 - SWISS-2DPAGE a database of two-dimensional gel electrophoresis images Electrophoresis 14 1232-1238
Appel RD Bairoch A Sanchez JC Vargas JR Golaz O Pasquali C and Hochstrasser DF 1996 ndash Federated two-dimensional electrophoresis database a simple means of publishing two-dimensional electrophoresis data Electrophoresis 17 540-546
Bjellqvist B Ek K Righetti PG Gianazza E Gorg A Westermeier R Postel W 1982 ndash Isoelectric focusing in immobilised pH gradients principle methodology and applications JBiochemBiophysMethods 6 317-339
Brazma A et al 2001 ndash Minimum information about a microarray experiment (MIAME)-towards standards for microarray data Nat genetics 29 365-71
Hoogland C Baujard Sanchez JC Hochstrasser DF and Appel RD 1997 ndash Make2ddb a simple package to set up a two-diensional electrophoresis database for the world wide web Electrophoresis 18 2755-2758
OFarrell 1975 - High resolution two-dimensional electrophoresis of proteins JBiolChem 25 250 4007-21
Vigorous lysis method
1 Sonication probe (cell suspension)
Avoid overheat cool on ice between burst
2 French pressure (microorganism with cell wall)
Cells are lysed by shear force
3 Mortar and pestle (solid tissue microorganism)
Grind solid tissue to fine powder with liquid nitrogen
4 Sample grinding kit (for small amount of sample)
For precious sample
5 Glass bead (cell suspension microorganism)
Using abrasive vortexed bead to break cell walls
Chaotrope
Urea (up to 9 M)
Thiourea (up to 2 M)
bull Disrupt of hydrogen and hydrophobic bonds
Note Urea (if tdeggt37degC) cyanate (HN=C=O)
carbamylation (Lys Arg)
(R-NH2 R-NH-CO-NH2)
carbamylation trains
Reductants
b-mercaptoethanol
DTT(dithiothreitol)
bull Break disulfide bridge
(within or between protein)
Detergents ndash surfactants
CHAPS (up to 4)
bull Disrupt membranes
bull Break hydrophobic interactions
bull Solubilise lipids
bull Release membrane bound proteins
Note No net charge for IEF (no SDS)
Soluble in urea
Isoelectric Focusing
Measure the protein concenteration in you samples
1 Biuret
2 Lowry methods
3 Bradford methods
4 UV methods
5 Special methods
6 Other commercial methods
BCA assay (bicinchoninic acid assay Pierce)
DC protein assay (detergent compatible Bio-rad)
DCRC protein assay (detergentreducing agent compatible
Bio-rad)
Before runninng IEF you shouldhellip
Introduced by Gorg A
Ref Gorg A (1994) Westermeier (2001)
Dried gel strips can be stored at -20 to -
80 from months to years
Amersham Biosciences Bio-Rad
IPG strips (3 mm x 18 cm x 05 mm)
Narrow range
Medium range
Broad range
4 7
35 45 55 67
40 50 60
3 10
6 11
Load 400 ml sample per groove (no bubbles)
Place IPG strip gel facing down in groove
Add ~25 ml non conductive oil per groove
Peel off protective film from strip
Rehydrate overnight (~22 hrs) at room temperature
Program power supply
bull Number of gels (1-10)
bull Max voltage 5000 V
bull Vhold 125 V
bull Duration 24 hrs 00 min
bull Max current 80 mAstrip
bull Volt hours 80000 Vh
pI
+
+ - - - - - -
+ + + + + + + -
pH 4 pH 7
Cathode Anode
Place wet wicks (DH2O) under each end of strip
Set chiller temperature for 20degC (setting ~25)
Remove IPG strip from tray
Let oil drip off the strip
Place IPG strip gel facing up in equilibration tray
+ -
Add 10 ml equilibration buffer 1 per tray
+ -
+ -
+ -
+ -
(in chemical hood)
MW 4 7
+ -
10 DuracrylTM gel (22 cm x 23 cm x 1 mm)
bull TrisTricineSDS buffer
bull IPG strip
bull MW (gel worm)
bull Agarose overlay
bull Silicone spacer
bull Gasket
-
+
Conditions
bull Number of gel (1-10)
bull Max voltage 500 V
bull Max power 1600 mWgel
bull 4degC (setting 10)
bull ~19 hrs
10 DuracrylTM gel (22 cm x 23 cm x 1 mm)
MW 4 7
+ -
Trisacetate 25 mM Tris-base pH 83 (acetic acid)
200 mM Tris-base 200 mM Tricine 04 SDS
045 mm filtered
TrisTricine SDS
Bio-Rad
Digest to peptide fragment MS analysis
1 First dimension
isoelectric focusing
separation according to the pI
2 Second dimension
SDS electrophoresis (SDS-PAGE)
Separation according to the MW
Interested spot
Sensitivity Process
TimeSteps
Advantages
bullBio-safe Coomassie 10ng 25hr3 steps MS compatible easily visualised non
hazardous
bullCoomassie Blue R-250 40ng 25hr2 steps Oldest and least expensive method
bullSilver Stain Plus 1ng 15hr3 steps MS compatible high sensitivity low
background
bullBio-Rad silver stain 1ng 2hr7 steps High sensitivity detects some highly
glycosylated and other difficult to
stain proteins
bullSypro ruby 1ng 3hr2 steps MS compatible allows analysis in
flourescent imagers linear over 3
orders of magnitude
Coomassie staining
bull moderate sensitivity (36-47 ng)
bull non specific
bull not quantitative
Silver staining
bull sensitive (05-12 ng)
bull time consuming
bull non specific
bull negative stain some spots
Fluorescent dye (SYPRO ruby)
bull sensitive (1-2 ng)
bull specific quantitative
bull end point stain
Coomassie Silver
Coomassie Silver Stain Copper Stain
SDS-polyacrylamide gel
Electrotransfer to membrane
Phosphoimager for 32P and 35S labelled
1D or 2D gels
Fluoroimager for
SYPRO labelled
1D or 2D gels
Densitometer or
Photo Scanner
Normal liver
Tumor
Both
Melanie 4 (GeneBio - Windows only)
httpcaexpasyorgmelanie
ImageMaster 2D Elite (Amersham)
httpwwwimsupportcom
Phoretix 2D Advanced
httpwwwphoretixcom
PDQuest 61 (BioRad - Windows only) httpwwwproteomeworksbio-radcomhtmlpdquesthtml
Mass Spectrometry
wwwexpasych - Swiss-2DPAGE httpwwwanlgovBIOPMG - Mouse liver human breast cell lines pyrococcus Argonne Protein Mapping Group httpwwwharefieldnthamesnhsuknhliproteinindexhtml - HSC-2DPAGE Heart Science Centre Harefield Hospital httpotowustleduthcperi-gelshtm - Washington Univ Inner Ear Protein Database
httpcaexpasyorgch2d2d-indexhtml - World 2DPAGE Index of 2D gel databases
Time line ~ 4 days
bull Protein gt 250 kDa do not enter 2 SDS-PAGE properly
bull PH more than 9
bull PH less than 4
bull Hydrophobic proteins
bull Low abundant proteins
Immunoblotting Proteins from 2D gels were transferred to PVDFmembranes The membranes were blocked with 5 skimmed milk in PBS-Tween (PBS-T) for 3 h The membranes were incubated overnight at 4degC with individual sera diluted 1100 in PBS-T followed by secondary antibody (anti-human IgG conjugated with horseradish peroxidase) for 2 h with dilutions of 13000 Before and after addition of the secondary antibody membranes washed 4 times each time for 5 min in PBS-T To visualize antigenic components membranes were incubated in diaminobenzeden (DAB) and H2O2 for 15 min
List of protein spots that reacted with more than 30 of serum samples
and were identified by mass spectrometry
1Heat shock protein HSP 90-beta
2 precursor(GRP 78) 78 kDa glucose-regulated protein
3 Heat shock cognate 71 kDa protein
4 Stress-70 protein mitochondrial precursor
5 Tubulin beta-5 chain
6Tubulin alpha-6 chain and 60 kDa heat shock protein mitochondrial
precursor (Hsp60)
7 Heterogeneous nuclear ribonucleoprotein K (hnRNP K)
8 Cytokeratin 8
9 Cytokeratin 18
10 Beta-actin
11 cytokeratin 19
12 Eukaryotic initiation factor 4A-I
13 14-3-3 protein epsilon
14 14-3-3 protein zetadelta (Protein kinase Cinhibitor protein-1)
15 Translationally controlled tumor protein (TCTP)
Among these autoantigens cytokeratin 8 cytokeratin 18 Fructose-bisphosphate aldolase and enolase were reacted with more than 60 of healthy individualsrsquo serum samples These antigens were intense spots in gels
Heat Shock proteins tubulins heterogeneous nuclear ribonucleoprotein actins cytokeratins 14-3-3 protein epsilon 14-3-3 protein zetadelta eukaryotic initiation factors Rho GDP-dissociation inhibitors phosphoglycerate mutase 1 G6PD GA3PD peroxiredoxins F-acting capping protein and triosephosphate isomerase were previously reported to induce humoral arm of the immune response in cancer
Translationally controlled tumor protein and electron transfer flavoprotein alpha-subunit have not been reported to induce humoral arm of the immune response in cancer so far
In terms of biological processes the identified proteins
belong to several groups
They are mainely involved in cell motility cytoskleton
stress response apoptosis cell signaling and
metabolism
Identification of other immunogenic proteins is
underway
Appel RD et al 1993 - SWISS-2DPAGE a database of two-dimensional gel electrophoresis images Electrophoresis 14 1232-1238
Appel RD Bairoch A Sanchez JC Vargas JR Golaz O Pasquali C and Hochstrasser DF 1996 ndash Federated two-dimensional electrophoresis database a simple means of publishing two-dimensional electrophoresis data Electrophoresis 17 540-546
Bjellqvist B Ek K Righetti PG Gianazza E Gorg A Westermeier R Postel W 1982 ndash Isoelectric focusing in immobilised pH gradients principle methodology and applications JBiochemBiophysMethods 6 317-339
Brazma A et al 2001 ndash Minimum information about a microarray experiment (MIAME)-towards standards for microarray data Nat genetics 29 365-71
Hoogland C Baujard Sanchez JC Hochstrasser DF and Appel RD 1997 ndash Make2ddb a simple package to set up a two-diensional electrophoresis database for the world wide web Electrophoresis 18 2755-2758
OFarrell 1975 - High resolution two-dimensional electrophoresis of proteins JBiolChem 25 250 4007-21
Chaotrope
Urea (up to 9 M)
Thiourea (up to 2 M)
bull Disrupt of hydrogen and hydrophobic bonds
Note Urea (if tdeggt37degC) cyanate (HN=C=O)
carbamylation (Lys Arg)
(R-NH2 R-NH-CO-NH2)
carbamylation trains
Reductants
b-mercaptoethanol
DTT(dithiothreitol)
bull Break disulfide bridge
(within or between protein)
Detergents ndash surfactants
CHAPS (up to 4)
bull Disrupt membranes
bull Break hydrophobic interactions
bull Solubilise lipids
bull Release membrane bound proteins
Note No net charge for IEF (no SDS)
Soluble in urea
Isoelectric Focusing
Measure the protein concenteration in you samples
1 Biuret
2 Lowry methods
3 Bradford methods
4 UV methods
5 Special methods
6 Other commercial methods
BCA assay (bicinchoninic acid assay Pierce)
DC protein assay (detergent compatible Bio-rad)
DCRC protein assay (detergentreducing agent compatible
Bio-rad)
Before runninng IEF you shouldhellip
Introduced by Gorg A
Ref Gorg A (1994) Westermeier (2001)
Dried gel strips can be stored at -20 to -
80 from months to years
Amersham Biosciences Bio-Rad
IPG strips (3 mm x 18 cm x 05 mm)
Narrow range
Medium range
Broad range
4 7
35 45 55 67
40 50 60
3 10
6 11
Load 400 ml sample per groove (no bubbles)
Place IPG strip gel facing down in groove
Add ~25 ml non conductive oil per groove
Peel off protective film from strip
Rehydrate overnight (~22 hrs) at room temperature
Program power supply
bull Number of gels (1-10)
bull Max voltage 5000 V
bull Vhold 125 V
bull Duration 24 hrs 00 min
bull Max current 80 mAstrip
bull Volt hours 80000 Vh
pI
+
+ - - - - - -
+ + + + + + + -
pH 4 pH 7
Cathode Anode
Place wet wicks (DH2O) under each end of strip
Set chiller temperature for 20degC (setting ~25)
Remove IPG strip from tray
Let oil drip off the strip
Place IPG strip gel facing up in equilibration tray
+ -
Add 10 ml equilibration buffer 1 per tray
+ -
+ -
+ -
+ -
(in chemical hood)
MW 4 7
+ -
10 DuracrylTM gel (22 cm x 23 cm x 1 mm)
bull TrisTricineSDS buffer
bull IPG strip
bull MW (gel worm)
bull Agarose overlay
bull Silicone spacer
bull Gasket
-
+
Conditions
bull Number of gel (1-10)
bull Max voltage 500 V
bull Max power 1600 mWgel
bull 4degC (setting 10)
bull ~19 hrs
10 DuracrylTM gel (22 cm x 23 cm x 1 mm)
MW 4 7
+ -
Trisacetate 25 mM Tris-base pH 83 (acetic acid)
200 mM Tris-base 200 mM Tricine 04 SDS
045 mm filtered
TrisTricine SDS
Bio-Rad
Digest to peptide fragment MS analysis
1 First dimension
isoelectric focusing
separation according to the pI
2 Second dimension
SDS electrophoresis (SDS-PAGE)
Separation according to the MW
Interested spot
Sensitivity Process
TimeSteps
Advantages
bullBio-safe Coomassie 10ng 25hr3 steps MS compatible easily visualised non
hazardous
bullCoomassie Blue R-250 40ng 25hr2 steps Oldest and least expensive method
bullSilver Stain Plus 1ng 15hr3 steps MS compatible high sensitivity low
background
bullBio-Rad silver stain 1ng 2hr7 steps High sensitivity detects some highly
glycosylated and other difficult to
stain proteins
bullSypro ruby 1ng 3hr2 steps MS compatible allows analysis in
flourescent imagers linear over 3
orders of magnitude
Coomassie staining
bull moderate sensitivity (36-47 ng)
bull non specific
bull not quantitative
Silver staining
bull sensitive (05-12 ng)
bull time consuming
bull non specific
bull negative stain some spots
Fluorescent dye (SYPRO ruby)
bull sensitive (1-2 ng)
bull specific quantitative
bull end point stain
Coomassie Silver
Coomassie Silver Stain Copper Stain
SDS-polyacrylamide gel
Electrotransfer to membrane
Phosphoimager for 32P and 35S labelled
1D or 2D gels
Fluoroimager for
SYPRO labelled
1D or 2D gels
Densitometer or
Photo Scanner
Normal liver
Tumor
Both
Melanie 4 (GeneBio - Windows only)
httpcaexpasyorgmelanie
ImageMaster 2D Elite (Amersham)
httpwwwimsupportcom
Phoretix 2D Advanced
httpwwwphoretixcom
PDQuest 61 (BioRad - Windows only) httpwwwproteomeworksbio-radcomhtmlpdquesthtml
Mass Spectrometry
wwwexpasych - Swiss-2DPAGE httpwwwanlgovBIOPMG - Mouse liver human breast cell lines pyrococcus Argonne Protein Mapping Group httpwwwharefieldnthamesnhsuknhliproteinindexhtml - HSC-2DPAGE Heart Science Centre Harefield Hospital httpotowustleduthcperi-gelshtm - Washington Univ Inner Ear Protein Database
httpcaexpasyorgch2d2d-indexhtml - World 2DPAGE Index of 2D gel databases
Time line ~ 4 days
bull Protein gt 250 kDa do not enter 2 SDS-PAGE properly
bull PH more than 9
bull PH less than 4
bull Hydrophobic proteins
bull Low abundant proteins
Immunoblotting Proteins from 2D gels were transferred to PVDFmembranes The membranes were blocked with 5 skimmed milk in PBS-Tween (PBS-T) for 3 h The membranes were incubated overnight at 4degC with individual sera diluted 1100 in PBS-T followed by secondary antibody (anti-human IgG conjugated with horseradish peroxidase) for 2 h with dilutions of 13000 Before and after addition of the secondary antibody membranes washed 4 times each time for 5 min in PBS-T To visualize antigenic components membranes were incubated in diaminobenzeden (DAB) and H2O2 for 15 min
List of protein spots that reacted with more than 30 of serum samples
and were identified by mass spectrometry
1Heat shock protein HSP 90-beta
2 precursor(GRP 78) 78 kDa glucose-regulated protein
3 Heat shock cognate 71 kDa protein
4 Stress-70 protein mitochondrial precursor
5 Tubulin beta-5 chain
6Tubulin alpha-6 chain and 60 kDa heat shock protein mitochondrial
precursor (Hsp60)
7 Heterogeneous nuclear ribonucleoprotein K (hnRNP K)
8 Cytokeratin 8
9 Cytokeratin 18
10 Beta-actin
11 cytokeratin 19
12 Eukaryotic initiation factor 4A-I
13 14-3-3 protein epsilon
14 14-3-3 protein zetadelta (Protein kinase Cinhibitor protein-1)
15 Translationally controlled tumor protein (TCTP)
Among these autoantigens cytokeratin 8 cytokeratin 18 Fructose-bisphosphate aldolase and enolase were reacted with more than 60 of healthy individualsrsquo serum samples These antigens were intense spots in gels
Heat Shock proteins tubulins heterogeneous nuclear ribonucleoprotein actins cytokeratins 14-3-3 protein epsilon 14-3-3 protein zetadelta eukaryotic initiation factors Rho GDP-dissociation inhibitors phosphoglycerate mutase 1 G6PD GA3PD peroxiredoxins F-acting capping protein and triosephosphate isomerase were previously reported to induce humoral arm of the immune response in cancer
Translationally controlled tumor protein and electron transfer flavoprotein alpha-subunit have not been reported to induce humoral arm of the immune response in cancer so far
In terms of biological processes the identified proteins
belong to several groups
They are mainely involved in cell motility cytoskleton
stress response apoptosis cell signaling and
metabolism
Identification of other immunogenic proteins is
underway
Appel RD et al 1993 - SWISS-2DPAGE a database of two-dimensional gel electrophoresis images Electrophoresis 14 1232-1238
Appel RD Bairoch A Sanchez JC Vargas JR Golaz O Pasquali C and Hochstrasser DF 1996 ndash Federated two-dimensional electrophoresis database a simple means of publishing two-dimensional electrophoresis data Electrophoresis 17 540-546
Bjellqvist B Ek K Righetti PG Gianazza E Gorg A Westermeier R Postel W 1982 ndash Isoelectric focusing in immobilised pH gradients principle methodology and applications JBiochemBiophysMethods 6 317-339
Brazma A et al 2001 ndash Minimum information about a microarray experiment (MIAME)-towards standards for microarray data Nat genetics 29 365-71
Hoogland C Baujard Sanchez JC Hochstrasser DF and Appel RD 1997 ndash Make2ddb a simple package to set up a two-diensional electrophoresis database for the world wide web Electrophoresis 18 2755-2758
OFarrell 1975 - High resolution two-dimensional electrophoresis of proteins JBiolChem 25 250 4007-21
Reductants
b-mercaptoethanol
DTT(dithiothreitol)
bull Break disulfide bridge
(within or between protein)
Detergents ndash surfactants
CHAPS (up to 4)
bull Disrupt membranes
bull Break hydrophobic interactions
bull Solubilise lipids
bull Release membrane bound proteins
Note No net charge for IEF (no SDS)
Soluble in urea
Isoelectric Focusing
Measure the protein concenteration in you samples
1 Biuret
2 Lowry methods
3 Bradford methods
4 UV methods
5 Special methods
6 Other commercial methods
BCA assay (bicinchoninic acid assay Pierce)
DC protein assay (detergent compatible Bio-rad)
DCRC protein assay (detergentreducing agent compatible
Bio-rad)
Before runninng IEF you shouldhellip
Introduced by Gorg A
Ref Gorg A (1994) Westermeier (2001)
Dried gel strips can be stored at -20 to -
80 from months to years
Amersham Biosciences Bio-Rad
IPG strips (3 mm x 18 cm x 05 mm)
Narrow range
Medium range
Broad range
4 7
35 45 55 67
40 50 60
3 10
6 11
Load 400 ml sample per groove (no bubbles)
Place IPG strip gel facing down in groove
Add ~25 ml non conductive oil per groove
Peel off protective film from strip
Rehydrate overnight (~22 hrs) at room temperature
Program power supply
bull Number of gels (1-10)
bull Max voltage 5000 V
bull Vhold 125 V
bull Duration 24 hrs 00 min
bull Max current 80 mAstrip
bull Volt hours 80000 Vh
pI
+
+ - - - - - -
+ + + + + + + -
pH 4 pH 7
Cathode Anode
Place wet wicks (DH2O) under each end of strip
Set chiller temperature for 20degC (setting ~25)
Remove IPG strip from tray
Let oil drip off the strip
Place IPG strip gel facing up in equilibration tray
+ -
Add 10 ml equilibration buffer 1 per tray
+ -
+ -
+ -
+ -
(in chemical hood)
MW 4 7
+ -
10 DuracrylTM gel (22 cm x 23 cm x 1 mm)
bull TrisTricineSDS buffer
bull IPG strip
bull MW (gel worm)
bull Agarose overlay
bull Silicone spacer
bull Gasket
-
+
Conditions
bull Number of gel (1-10)
bull Max voltage 500 V
bull Max power 1600 mWgel
bull 4degC (setting 10)
bull ~19 hrs
10 DuracrylTM gel (22 cm x 23 cm x 1 mm)
MW 4 7
+ -
Trisacetate 25 mM Tris-base pH 83 (acetic acid)
200 mM Tris-base 200 mM Tricine 04 SDS
045 mm filtered
TrisTricine SDS
Bio-Rad
Digest to peptide fragment MS analysis
1 First dimension
isoelectric focusing
separation according to the pI
2 Second dimension
SDS electrophoresis (SDS-PAGE)
Separation according to the MW
Interested spot
Sensitivity Process
TimeSteps
Advantages
bullBio-safe Coomassie 10ng 25hr3 steps MS compatible easily visualised non
hazardous
bullCoomassie Blue R-250 40ng 25hr2 steps Oldest and least expensive method
bullSilver Stain Plus 1ng 15hr3 steps MS compatible high sensitivity low
background
bullBio-Rad silver stain 1ng 2hr7 steps High sensitivity detects some highly
glycosylated and other difficult to
stain proteins
bullSypro ruby 1ng 3hr2 steps MS compatible allows analysis in
flourescent imagers linear over 3
orders of magnitude
Coomassie staining
bull moderate sensitivity (36-47 ng)
bull non specific
bull not quantitative
Silver staining
bull sensitive (05-12 ng)
bull time consuming
bull non specific
bull negative stain some spots
Fluorescent dye (SYPRO ruby)
bull sensitive (1-2 ng)
bull specific quantitative
bull end point stain
Coomassie Silver
Coomassie Silver Stain Copper Stain
SDS-polyacrylamide gel
Electrotransfer to membrane
Phosphoimager for 32P and 35S labelled
1D or 2D gels
Fluoroimager for
SYPRO labelled
1D or 2D gels
Densitometer or
Photo Scanner
Normal liver
Tumor
Both
Melanie 4 (GeneBio - Windows only)
httpcaexpasyorgmelanie
ImageMaster 2D Elite (Amersham)
httpwwwimsupportcom
Phoretix 2D Advanced
httpwwwphoretixcom
PDQuest 61 (BioRad - Windows only) httpwwwproteomeworksbio-radcomhtmlpdquesthtml
Mass Spectrometry
wwwexpasych - Swiss-2DPAGE httpwwwanlgovBIOPMG - Mouse liver human breast cell lines pyrococcus Argonne Protein Mapping Group httpwwwharefieldnthamesnhsuknhliproteinindexhtml - HSC-2DPAGE Heart Science Centre Harefield Hospital httpotowustleduthcperi-gelshtm - Washington Univ Inner Ear Protein Database
httpcaexpasyorgch2d2d-indexhtml - World 2DPAGE Index of 2D gel databases
Time line ~ 4 days
bull Protein gt 250 kDa do not enter 2 SDS-PAGE properly
bull PH more than 9
bull PH less than 4
bull Hydrophobic proteins
bull Low abundant proteins
Immunoblotting Proteins from 2D gels were transferred to PVDFmembranes The membranes were blocked with 5 skimmed milk in PBS-Tween (PBS-T) for 3 h The membranes were incubated overnight at 4degC with individual sera diluted 1100 in PBS-T followed by secondary antibody (anti-human IgG conjugated with horseradish peroxidase) for 2 h with dilutions of 13000 Before and after addition of the secondary antibody membranes washed 4 times each time for 5 min in PBS-T To visualize antigenic components membranes were incubated in diaminobenzeden (DAB) and H2O2 for 15 min
List of protein spots that reacted with more than 30 of serum samples
and were identified by mass spectrometry
1Heat shock protein HSP 90-beta
2 precursor(GRP 78) 78 kDa glucose-regulated protein
3 Heat shock cognate 71 kDa protein
4 Stress-70 protein mitochondrial precursor
5 Tubulin beta-5 chain
6Tubulin alpha-6 chain and 60 kDa heat shock protein mitochondrial
precursor (Hsp60)
7 Heterogeneous nuclear ribonucleoprotein K (hnRNP K)
8 Cytokeratin 8
9 Cytokeratin 18
10 Beta-actin
11 cytokeratin 19
12 Eukaryotic initiation factor 4A-I
13 14-3-3 protein epsilon
14 14-3-3 protein zetadelta (Protein kinase Cinhibitor protein-1)
15 Translationally controlled tumor protein (TCTP)
Among these autoantigens cytokeratin 8 cytokeratin 18 Fructose-bisphosphate aldolase and enolase were reacted with more than 60 of healthy individualsrsquo serum samples These antigens were intense spots in gels
Heat Shock proteins tubulins heterogeneous nuclear ribonucleoprotein actins cytokeratins 14-3-3 protein epsilon 14-3-3 protein zetadelta eukaryotic initiation factors Rho GDP-dissociation inhibitors phosphoglycerate mutase 1 G6PD GA3PD peroxiredoxins F-acting capping protein and triosephosphate isomerase were previously reported to induce humoral arm of the immune response in cancer
Translationally controlled tumor protein and electron transfer flavoprotein alpha-subunit have not been reported to induce humoral arm of the immune response in cancer so far
In terms of biological processes the identified proteins
belong to several groups
They are mainely involved in cell motility cytoskleton
stress response apoptosis cell signaling and
metabolism
Identification of other immunogenic proteins is
underway
Appel RD et al 1993 - SWISS-2DPAGE a database of two-dimensional gel electrophoresis images Electrophoresis 14 1232-1238
Appel RD Bairoch A Sanchez JC Vargas JR Golaz O Pasquali C and Hochstrasser DF 1996 ndash Federated two-dimensional electrophoresis database a simple means of publishing two-dimensional electrophoresis data Electrophoresis 17 540-546
Bjellqvist B Ek K Righetti PG Gianazza E Gorg A Westermeier R Postel W 1982 ndash Isoelectric focusing in immobilised pH gradients principle methodology and applications JBiochemBiophysMethods 6 317-339
Brazma A et al 2001 ndash Minimum information about a microarray experiment (MIAME)-towards standards for microarray data Nat genetics 29 365-71
Hoogland C Baujard Sanchez JC Hochstrasser DF and Appel RD 1997 ndash Make2ddb a simple package to set up a two-diensional electrophoresis database for the world wide web Electrophoresis 18 2755-2758
OFarrell 1975 - High resolution two-dimensional electrophoresis of proteins JBiolChem 25 250 4007-21
Detergents ndash surfactants
CHAPS (up to 4)
bull Disrupt membranes
bull Break hydrophobic interactions
bull Solubilise lipids
bull Release membrane bound proteins
Note No net charge for IEF (no SDS)
Soluble in urea
Isoelectric Focusing
Measure the protein concenteration in you samples
1 Biuret
2 Lowry methods
3 Bradford methods
4 UV methods
5 Special methods
6 Other commercial methods
BCA assay (bicinchoninic acid assay Pierce)
DC protein assay (detergent compatible Bio-rad)
DCRC protein assay (detergentreducing agent compatible
Bio-rad)
Before runninng IEF you shouldhellip
Introduced by Gorg A
Ref Gorg A (1994) Westermeier (2001)
Dried gel strips can be stored at -20 to -
80 from months to years
Amersham Biosciences Bio-Rad
IPG strips (3 mm x 18 cm x 05 mm)
Narrow range
Medium range
Broad range
4 7
35 45 55 67
40 50 60
3 10
6 11
Load 400 ml sample per groove (no bubbles)
Place IPG strip gel facing down in groove
Add ~25 ml non conductive oil per groove
Peel off protective film from strip
Rehydrate overnight (~22 hrs) at room temperature
Program power supply
bull Number of gels (1-10)
bull Max voltage 5000 V
bull Vhold 125 V
bull Duration 24 hrs 00 min
bull Max current 80 mAstrip
bull Volt hours 80000 Vh
pI
+
+ - - - - - -
+ + + + + + + -
pH 4 pH 7
Cathode Anode
Place wet wicks (DH2O) under each end of strip
Set chiller temperature for 20degC (setting ~25)
Remove IPG strip from tray
Let oil drip off the strip
Place IPG strip gel facing up in equilibration tray
+ -
Add 10 ml equilibration buffer 1 per tray
+ -
+ -
+ -
+ -
(in chemical hood)
MW 4 7
+ -
10 DuracrylTM gel (22 cm x 23 cm x 1 mm)
bull TrisTricineSDS buffer
bull IPG strip
bull MW (gel worm)
bull Agarose overlay
bull Silicone spacer
bull Gasket
-
+
Conditions
bull Number of gel (1-10)
bull Max voltage 500 V
bull Max power 1600 mWgel
bull 4degC (setting 10)
bull ~19 hrs
10 DuracrylTM gel (22 cm x 23 cm x 1 mm)
MW 4 7
+ -
Trisacetate 25 mM Tris-base pH 83 (acetic acid)
200 mM Tris-base 200 mM Tricine 04 SDS
045 mm filtered
TrisTricine SDS
Bio-Rad
Digest to peptide fragment MS analysis
1 First dimension
isoelectric focusing
separation according to the pI
2 Second dimension
SDS electrophoresis (SDS-PAGE)
Separation according to the MW
Interested spot
Sensitivity Process
TimeSteps
Advantages
bullBio-safe Coomassie 10ng 25hr3 steps MS compatible easily visualised non
hazardous
bullCoomassie Blue R-250 40ng 25hr2 steps Oldest and least expensive method
bullSilver Stain Plus 1ng 15hr3 steps MS compatible high sensitivity low
background
bullBio-Rad silver stain 1ng 2hr7 steps High sensitivity detects some highly
glycosylated and other difficult to
stain proteins
bullSypro ruby 1ng 3hr2 steps MS compatible allows analysis in
flourescent imagers linear over 3
orders of magnitude
Coomassie staining
bull moderate sensitivity (36-47 ng)
bull non specific
bull not quantitative
Silver staining
bull sensitive (05-12 ng)
bull time consuming
bull non specific
bull negative stain some spots
Fluorescent dye (SYPRO ruby)
bull sensitive (1-2 ng)
bull specific quantitative
bull end point stain
Coomassie Silver
Coomassie Silver Stain Copper Stain
SDS-polyacrylamide gel
Electrotransfer to membrane
Phosphoimager for 32P and 35S labelled
1D or 2D gels
Fluoroimager for
SYPRO labelled
1D or 2D gels
Densitometer or
Photo Scanner
Normal liver
Tumor
Both
Melanie 4 (GeneBio - Windows only)
httpcaexpasyorgmelanie
ImageMaster 2D Elite (Amersham)
httpwwwimsupportcom
Phoretix 2D Advanced
httpwwwphoretixcom
PDQuest 61 (BioRad - Windows only) httpwwwproteomeworksbio-radcomhtmlpdquesthtml
Mass Spectrometry
wwwexpasych - Swiss-2DPAGE httpwwwanlgovBIOPMG - Mouse liver human breast cell lines pyrococcus Argonne Protein Mapping Group httpwwwharefieldnthamesnhsuknhliproteinindexhtml - HSC-2DPAGE Heart Science Centre Harefield Hospital httpotowustleduthcperi-gelshtm - Washington Univ Inner Ear Protein Database
httpcaexpasyorgch2d2d-indexhtml - World 2DPAGE Index of 2D gel databases
Time line ~ 4 days
bull Protein gt 250 kDa do not enter 2 SDS-PAGE properly
bull PH more than 9
bull PH less than 4
bull Hydrophobic proteins
bull Low abundant proteins
Immunoblotting Proteins from 2D gels were transferred to PVDFmembranes The membranes were blocked with 5 skimmed milk in PBS-Tween (PBS-T) for 3 h The membranes were incubated overnight at 4degC with individual sera diluted 1100 in PBS-T followed by secondary antibody (anti-human IgG conjugated with horseradish peroxidase) for 2 h with dilutions of 13000 Before and after addition of the secondary antibody membranes washed 4 times each time for 5 min in PBS-T To visualize antigenic components membranes were incubated in diaminobenzeden (DAB) and H2O2 for 15 min
List of protein spots that reacted with more than 30 of serum samples
and were identified by mass spectrometry
1Heat shock protein HSP 90-beta
2 precursor(GRP 78) 78 kDa glucose-regulated protein
3 Heat shock cognate 71 kDa protein
4 Stress-70 protein mitochondrial precursor
5 Tubulin beta-5 chain
6Tubulin alpha-6 chain and 60 kDa heat shock protein mitochondrial
precursor (Hsp60)
7 Heterogeneous nuclear ribonucleoprotein K (hnRNP K)
8 Cytokeratin 8
9 Cytokeratin 18
10 Beta-actin
11 cytokeratin 19
12 Eukaryotic initiation factor 4A-I
13 14-3-3 protein epsilon
14 14-3-3 protein zetadelta (Protein kinase Cinhibitor protein-1)
15 Translationally controlled tumor protein (TCTP)
Among these autoantigens cytokeratin 8 cytokeratin 18 Fructose-bisphosphate aldolase and enolase were reacted with more than 60 of healthy individualsrsquo serum samples These antigens were intense spots in gels
Heat Shock proteins tubulins heterogeneous nuclear ribonucleoprotein actins cytokeratins 14-3-3 protein epsilon 14-3-3 protein zetadelta eukaryotic initiation factors Rho GDP-dissociation inhibitors phosphoglycerate mutase 1 G6PD GA3PD peroxiredoxins F-acting capping protein and triosephosphate isomerase were previously reported to induce humoral arm of the immune response in cancer
Translationally controlled tumor protein and electron transfer flavoprotein alpha-subunit have not been reported to induce humoral arm of the immune response in cancer so far
In terms of biological processes the identified proteins
belong to several groups
They are mainely involved in cell motility cytoskleton
stress response apoptosis cell signaling and
metabolism
Identification of other immunogenic proteins is
underway
Appel RD et al 1993 - SWISS-2DPAGE a database of two-dimensional gel electrophoresis images Electrophoresis 14 1232-1238
Appel RD Bairoch A Sanchez JC Vargas JR Golaz O Pasquali C and Hochstrasser DF 1996 ndash Federated two-dimensional electrophoresis database a simple means of publishing two-dimensional electrophoresis data Electrophoresis 17 540-546
Bjellqvist B Ek K Righetti PG Gianazza E Gorg A Westermeier R Postel W 1982 ndash Isoelectric focusing in immobilised pH gradients principle methodology and applications JBiochemBiophysMethods 6 317-339
Brazma A et al 2001 ndash Minimum information about a microarray experiment (MIAME)-towards standards for microarray data Nat genetics 29 365-71
Hoogland C Baujard Sanchez JC Hochstrasser DF and Appel RD 1997 ndash Make2ddb a simple package to set up a two-diensional electrophoresis database for the world wide web Electrophoresis 18 2755-2758
OFarrell 1975 - High resolution two-dimensional electrophoresis of proteins JBiolChem 25 250 4007-21
Isoelectric Focusing
Measure the protein concenteration in you samples
1 Biuret
2 Lowry methods
3 Bradford methods
4 UV methods
5 Special methods
6 Other commercial methods
BCA assay (bicinchoninic acid assay Pierce)
DC protein assay (detergent compatible Bio-rad)
DCRC protein assay (detergentreducing agent compatible
Bio-rad)
Before runninng IEF you shouldhellip
Introduced by Gorg A
Ref Gorg A (1994) Westermeier (2001)
Dried gel strips can be stored at -20 to -
80 from months to years
Amersham Biosciences Bio-Rad
IPG strips (3 mm x 18 cm x 05 mm)
Narrow range
Medium range
Broad range
4 7
35 45 55 67
40 50 60
3 10
6 11
Load 400 ml sample per groove (no bubbles)
Place IPG strip gel facing down in groove
Add ~25 ml non conductive oil per groove
Peel off protective film from strip
Rehydrate overnight (~22 hrs) at room temperature
Program power supply
bull Number of gels (1-10)
bull Max voltage 5000 V
bull Vhold 125 V
bull Duration 24 hrs 00 min
bull Max current 80 mAstrip
bull Volt hours 80000 Vh
pI
+
+ - - - - - -
+ + + + + + + -
pH 4 pH 7
Cathode Anode
Place wet wicks (DH2O) under each end of strip
Set chiller temperature for 20degC (setting ~25)
Remove IPG strip from tray
Let oil drip off the strip
Place IPG strip gel facing up in equilibration tray
+ -
Add 10 ml equilibration buffer 1 per tray
+ -
+ -
+ -
+ -
(in chemical hood)
MW 4 7
+ -
10 DuracrylTM gel (22 cm x 23 cm x 1 mm)
bull TrisTricineSDS buffer
bull IPG strip
bull MW (gel worm)
bull Agarose overlay
bull Silicone spacer
bull Gasket
-
+
Conditions
bull Number of gel (1-10)
bull Max voltage 500 V
bull Max power 1600 mWgel
bull 4degC (setting 10)
bull ~19 hrs
10 DuracrylTM gel (22 cm x 23 cm x 1 mm)
MW 4 7
+ -
Trisacetate 25 mM Tris-base pH 83 (acetic acid)
200 mM Tris-base 200 mM Tricine 04 SDS
045 mm filtered
TrisTricine SDS
Bio-Rad
Digest to peptide fragment MS analysis
1 First dimension
isoelectric focusing
separation according to the pI
2 Second dimension
SDS electrophoresis (SDS-PAGE)
Separation according to the MW
Interested spot
Sensitivity Process
TimeSteps
Advantages
bullBio-safe Coomassie 10ng 25hr3 steps MS compatible easily visualised non
hazardous
bullCoomassie Blue R-250 40ng 25hr2 steps Oldest and least expensive method
bullSilver Stain Plus 1ng 15hr3 steps MS compatible high sensitivity low
background
bullBio-Rad silver stain 1ng 2hr7 steps High sensitivity detects some highly
glycosylated and other difficult to
stain proteins
bullSypro ruby 1ng 3hr2 steps MS compatible allows analysis in
flourescent imagers linear over 3
orders of magnitude
Coomassie staining
bull moderate sensitivity (36-47 ng)
bull non specific
bull not quantitative
Silver staining
bull sensitive (05-12 ng)
bull time consuming
bull non specific
bull negative stain some spots
Fluorescent dye (SYPRO ruby)
bull sensitive (1-2 ng)
bull specific quantitative
bull end point stain
Coomassie Silver
Coomassie Silver Stain Copper Stain
SDS-polyacrylamide gel
Electrotransfer to membrane
Phosphoimager for 32P and 35S labelled
1D or 2D gels
Fluoroimager for
SYPRO labelled
1D or 2D gels
Densitometer or
Photo Scanner
Normal liver
Tumor
Both
Melanie 4 (GeneBio - Windows only)
httpcaexpasyorgmelanie
ImageMaster 2D Elite (Amersham)
httpwwwimsupportcom
Phoretix 2D Advanced
httpwwwphoretixcom
PDQuest 61 (BioRad - Windows only) httpwwwproteomeworksbio-radcomhtmlpdquesthtml
Mass Spectrometry
wwwexpasych - Swiss-2DPAGE httpwwwanlgovBIOPMG - Mouse liver human breast cell lines pyrococcus Argonne Protein Mapping Group httpwwwharefieldnthamesnhsuknhliproteinindexhtml - HSC-2DPAGE Heart Science Centre Harefield Hospital httpotowustleduthcperi-gelshtm - Washington Univ Inner Ear Protein Database
httpcaexpasyorgch2d2d-indexhtml - World 2DPAGE Index of 2D gel databases
Time line ~ 4 days
bull Protein gt 250 kDa do not enter 2 SDS-PAGE properly
bull PH more than 9
bull PH less than 4
bull Hydrophobic proteins
bull Low abundant proteins
Immunoblotting Proteins from 2D gels were transferred to PVDFmembranes The membranes were blocked with 5 skimmed milk in PBS-Tween (PBS-T) for 3 h The membranes were incubated overnight at 4degC with individual sera diluted 1100 in PBS-T followed by secondary antibody (anti-human IgG conjugated with horseradish peroxidase) for 2 h with dilutions of 13000 Before and after addition of the secondary antibody membranes washed 4 times each time for 5 min in PBS-T To visualize antigenic components membranes were incubated in diaminobenzeden (DAB) and H2O2 for 15 min
List of protein spots that reacted with more than 30 of serum samples
and were identified by mass spectrometry
1Heat shock protein HSP 90-beta
2 precursor(GRP 78) 78 kDa glucose-regulated protein
3 Heat shock cognate 71 kDa protein
4 Stress-70 protein mitochondrial precursor
5 Tubulin beta-5 chain
6Tubulin alpha-6 chain and 60 kDa heat shock protein mitochondrial
precursor (Hsp60)
7 Heterogeneous nuclear ribonucleoprotein K (hnRNP K)
8 Cytokeratin 8
9 Cytokeratin 18
10 Beta-actin
11 cytokeratin 19
12 Eukaryotic initiation factor 4A-I
13 14-3-3 protein epsilon
14 14-3-3 protein zetadelta (Protein kinase Cinhibitor protein-1)
15 Translationally controlled tumor protein (TCTP)
Among these autoantigens cytokeratin 8 cytokeratin 18 Fructose-bisphosphate aldolase and enolase were reacted with more than 60 of healthy individualsrsquo serum samples These antigens were intense spots in gels
Heat Shock proteins tubulins heterogeneous nuclear ribonucleoprotein actins cytokeratins 14-3-3 protein epsilon 14-3-3 protein zetadelta eukaryotic initiation factors Rho GDP-dissociation inhibitors phosphoglycerate mutase 1 G6PD GA3PD peroxiredoxins F-acting capping protein and triosephosphate isomerase were previously reported to induce humoral arm of the immune response in cancer
Translationally controlled tumor protein and electron transfer flavoprotein alpha-subunit have not been reported to induce humoral arm of the immune response in cancer so far
In terms of biological processes the identified proteins
belong to several groups
They are mainely involved in cell motility cytoskleton
stress response apoptosis cell signaling and
metabolism
Identification of other immunogenic proteins is
underway
Appel RD et al 1993 - SWISS-2DPAGE a database of two-dimensional gel electrophoresis images Electrophoresis 14 1232-1238
Appel RD Bairoch A Sanchez JC Vargas JR Golaz O Pasquali C and Hochstrasser DF 1996 ndash Federated two-dimensional electrophoresis database a simple means of publishing two-dimensional electrophoresis data Electrophoresis 17 540-546
Bjellqvist B Ek K Righetti PG Gianazza E Gorg A Westermeier R Postel W 1982 ndash Isoelectric focusing in immobilised pH gradients principle methodology and applications JBiochemBiophysMethods 6 317-339
Brazma A et al 2001 ndash Minimum information about a microarray experiment (MIAME)-towards standards for microarray data Nat genetics 29 365-71
Hoogland C Baujard Sanchez JC Hochstrasser DF and Appel RD 1997 ndash Make2ddb a simple package to set up a two-diensional electrophoresis database for the world wide web Electrophoresis 18 2755-2758
OFarrell 1975 - High resolution two-dimensional electrophoresis of proteins JBiolChem 25 250 4007-21
Measure the protein concenteration in you samples
1 Biuret
2 Lowry methods
3 Bradford methods
4 UV methods
5 Special methods
6 Other commercial methods
BCA assay (bicinchoninic acid assay Pierce)
DC protein assay (detergent compatible Bio-rad)
DCRC protein assay (detergentreducing agent compatible
Bio-rad)
Before runninng IEF you shouldhellip
Introduced by Gorg A
Ref Gorg A (1994) Westermeier (2001)
Dried gel strips can be stored at -20 to -
80 from months to years
Amersham Biosciences Bio-Rad
IPG strips (3 mm x 18 cm x 05 mm)
Narrow range
Medium range
Broad range
4 7
35 45 55 67
40 50 60
3 10
6 11
Load 400 ml sample per groove (no bubbles)
Place IPG strip gel facing down in groove
Add ~25 ml non conductive oil per groove
Peel off protective film from strip
Rehydrate overnight (~22 hrs) at room temperature
Program power supply
bull Number of gels (1-10)
bull Max voltage 5000 V
bull Vhold 125 V
bull Duration 24 hrs 00 min
bull Max current 80 mAstrip
bull Volt hours 80000 Vh
pI
+
+ - - - - - -
+ + + + + + + -
pH 4 pH 7
Cathode Anode
Place wet wicks (DH2O) under each end of strip
Set chiller temperature for 20degC (setting ~25)
Remove IPG strip from tray
Let oil drip off the strip
Place IPG strip gel facing up in equilibration tray
+ -
Add 10 ml equilibration buffer 1 per tray
+ -
+ -
+ -
+ -
(in chemical hood)
MW 4 7
+ -
10 DuracrylTM gel (22 cm x 23 cm x 1 mm)
bull TrisTricineSDS buffer
bull IPG strip
bull MW (gel worm)
bull Agarose overlay
bull Silicone spacer
bull Gasket
-
+
Conditions
bull Number of gel (1-10)
bull Max voltage 500 V
bull Max power 1600 mWgel
bull 4degC (setting 10)
bull ~19 hrs
10 DuracrylTM gel (22 cm x 23 cm x 1 mm)
MW 4 7
+ -
Trisacetate 25 mM Tris-base pH 83 (acetic acid)
200 mM Tris-base 200 mM Tricine 04 SDS
045 mm filtered
TrisTricine SDS
Bio-Rad
Digest to peptide fragment MS analysis
1 First dimension
isoelectric focusing
separation according to the pI
2 Second dimension
SDS electrophoresis (SDS-PAGE)
Separation according to the MW
Interested spot
Sensitivity Process
TimeSteps
Advantages
bullBio-safe Coomassie 10ng 25hr3 steps MS compatible easily visualised non
hazardous
bullCoomassie Blue R-250 40ng 25hr2 steps Oldest and least expensive method
bullSilver Stain Plus 1ng 15hr3 steps MS compatible high sensitivity low
background
bullBio-Rad silver stain 1ng 2hr7 steps High sensitivity detects some highly
glycosylated and other difficult to
stain proteins
bullSypro ruby 1ng 3hr2 steps MS compatible allows analysis in
flourescent imagers linear over 3
orders of magnitude
Coomassie staining
bull moderate sensitivity (36-47 ng)
bull non specific
bull not quantitative
Silver staining
bull sensitive (05-12 ng)
bull time consuming
bull non specific
bull negative stain some spots
Fluorescent dye (SYPRO ruby)
bull sensitive (1-2 ng)
bull specific quantitative
bull end point stain
Coomassie Silver
Coomassie Silver Stain Copper Stain
SDS-polyacrylamide gel
Electrotransfer to membrane
Phosphoimager for 32P and 35S labelled
1D or 2D gels
Fluoroimager for
SYPRO labelled
1D or 2D gels
Densitometer or
Photo Scanner
Normal liver
Tumor
Both
Melanie 4 (GeneBio - Windows only)
httpcaexpasyorgmelanie
ImageMaster 2D Elite (Amersham)
httpwwwimsupportcom
Phoretix 2D Advanced
httpwwwphoretixcom
PDQuest 61 (BioRad - Windows only) httpwwwproteomeworksbio-radcomhtmlpdquesthtml
Mass Spectrometry
wwwexpasych - Swiss-2DPAGE httpwwwanlgovBIOPMG - Mouse liver human breast cell lines pyrococcus Argonne Protein Mapping Group httpwwwharefieldnthamesnhsuknhliproteinindexhtml - HSC-2DPAGE Heart Science Centre Harefield Hospital httpotowustleduthcperi-gelshtm - Washington Univ Inner Ear Protein Database
httpcaexpasyorgch2d2d-indexhtml - World 2DPAGE Index of 2D gel databases
Time line ~ 4 days
bull Protein gt 250 kDa do not enter 2 SDS-PAGE properly
bull PH more than 9
bull PH less than 4
bull Hydrophobic proteins
bull Low abundant proteins
Immunoblotting Proteins from 2D gels were transferred to PVDFmembranes The membranes were blocked with 5 skimmed milk in PBS-Tween (PBS-T) for 3 h The membranes were incubated overnight at 4degC with individual sera diluted 1100 in PBS-T followed by secondary antibody (anti-human IgG conjugated with horseradish peroxidase) for 2 h with dilutions of 13000 Before and after addition of the secondary antibody membranes washed 4 times each time for 5 min in PBS-T To visualize antigenic components membranes were incubated in diaminobenzeden (DAB) and H2O2 for 15 min
List of protein spots that reacted with more than 30 of serum samples
and were identified by mass spectrometry
1Heat shock protein HSP 90-beta
2 precursor(GRP 78) 78 kDa glucose-regulated protein
3 Heat shock cognate 71 kDa protein
4 Stress-70 protein mitochondrial precursor
5 Tubulin beta-5 chain
6Tubulin alpha-6 chain and 60 kDa heat shock protein mitochondrial
precursor (Hsp60)
7 Heterogeneous nuclear ribonucleoprotein K (hnRNP K)
8 Cytokeratin 8
9 Cytokeratin 18
10 Beta-actin
11 cytokeratin 19
12 Eukaryotic initiation factor 4A-I
13 14-3-3 protein epsilon
14 14-3-3 protein zetadelta (Protein kinase Cinhibitor protein-1)
15 Translationally controlled tumor protein (TCTP)
Among these autoantigens cytokeratin 8 cytokeratin 18 Fructose-bisphosphate aldolase and enolase were reacted with more than 60 of healthy individualsrsquo serum samples These antigens were intense spots in gels
Heat Shock proteins tubulins heterogeneous nuclear ribonucleoprotein actins cytokeratins 14-3-3 protein epsilon 14-3-3 protein zetadelta eukaryotic initiation factors Rho GDP-dissociation inhibitors phosphoglycerate mutase 1 G6PD GA3PD peroxiredoxins F-acting capping protein and triosephosphate isomerase were previously reported to induce humoral arm of the immune response in cancer
Translationally controlled tumor protein and electron transfer flavoprotein alpha-subunit have not been reported to induce humoral arm of the immune response in cancer so far
In terms of biological processes the identified proteins
belong to several groups
They are mainely involved in cell motility cytoskleton
stress response apoptosis cell signaling and
metabolism
Identification of other immunogenic proteins is
underway
Appel RD et al 1993 - SWISS-2DPAGE a database of two-dimensional gel electrophoresis images Electrophoresis 14 1232-1238
Appel RD Bairoch A Sanchez JC Vargas JR Golaz O Pasquali C and Hochstrasser DF 1996 ndash Federated two-dimensional electrophoresis database a simple means of publishing two-dimensional electrophoresis data Electrophoresis 17 540-546
Bjellqvist B Ek K Righetti PG Gianazza E Gorg A Westermeier R Postel W 1982 ndash Isoelectric focusing in immobilised pH gradients principle methodology and applications JBiochemBiophysMethods 6 317-339
Brazma A et al 2001 ndash Minimum information about a microarray experiment (MIAME)-towards standards for microarray data Nat genetics 29 365-71
Hoogland C Baujard Sanchez JC Hochstrasser DF and Appel RD 1997 ndash Make2ddb a simple package to set up a two-diensional electrophoresis database for the world wide web Electrophoresis 18 2755-2758
OFarrell 1975 - High resolution two-dimensional electrophoresis of proteins JBiolChem 25 250 4007-21
Introduced by Gorg A
Ref Gorg A (1994) Westermeier (2001)
Dried gel strips can be stored at -20 to -
80 from months to years
Amersham Biosciences Bio-Rad
IPG strips (3 mm x 18 cm x 05 mm)
Narrow range
Medium range
Broad range
4 7
35 45 55 67
40 50 60
3 10
6 11
Load 400 ml sample per groove (no bubbles)
Place IPG strip gel facing down in groove
Add ~25 ml non conductive oil per groove
Peel off protective film from strip
Rehydrate overnight (~22 hrs) at room temperature
Program power supply
bull Number of gels (1-10)
bull Max voltage 5000 V
bull Vhold 125 V
bull Duration 24 hrs 00 min
bull Max current 80 mAstrip
bull Volt hours 80000 Vh
pI
+
+ - - - - - -
+ + + + + + + -
pH 4 pH 7
Cathode Anode
Place wet wicks (DH2O) under each end of strip
Set chiller temperature for 20degC (setting ~25)
Remove IPG strip from tray
Let oil drip off the strip
Place IPG strip gel facing up in equilibration tray
+ -
Add 10 ml equilibration buffer 1 per tray
+ -
+ -
+ -
+ -
(in chemical hood)
MW 4 7
+ -
10 DuracrylTM gel (22 cm x 23 cm x 1 mm)
bull TrisTricineSDS buffer
bull IPG strip
bull MW (gel worm)
bull Agarose overlay
bull Silicone spacer
bull Gasket
-
+
Conditions
bull Number of gel (1-10)
bull Max voltage 500 V
bull Max power 1600 mWgel
bull 4degC (setting 10)
bull ~19 hrs
10 DuracrylTM gel (22 cm x 23 cm x 1 mm)
MW 4 7
+ -
Trisacetate 25 mM Tris-base pH 83 (acetic acid)
200 mM Tris-base 200 mM Tricine 04 SDS
045 mm filtered
TrisTricine SDS
Bio-Rad
Digest to peptide fragment MS analysis
1 First dimension
isoelectric focusing
separation according to the pI
2 Second dimension
SDS electrophoresis (SDS-PAGE)
Separation according to the MW
Interested spot
Sensitivity Process
TimeSteps
Advantages
bullBio-safe Coomassie 10ng 25hr3 steps MS compatible easily visualised non
hazardous
bullCoomassie Blue R-250 40ng 25hr2 steps Oldest and least expensive method
bullSilver Stain Plus 1ng 15hr3 steps MS compatible high sensitivity low
background
bullBio-Rad silver stain 1ng 2hr7 steps High sensitivity detects some highly
glycosylated and other difficult to
stain proteins
bullSypro ruby 1ng 3hr2 steps MS compatible allows analysis in
flourescent imagers linear over 3
orders of magnitude
Coomassie staining
bull moderate sensitivity (36-47 ng)
bull non specific
bull not quantitative
Silver staining
bull sensitive (05-12 ng)
bull time consuming
bull non specific
bull negative stain some spots
Fluorescent dye (SYPRO ruby)
bull sensitive (1-2 ng)
bull specific quantitative
bull end point stain
Coomassie Silver
Coomassie Silver Stain Copper Stain
SDS-polyacrylamide gel
Electrotransfer to membrane
Phosphoimager for 32P and 35S labelled
1D or 2D gels
Fluoroimager for
SYPRO labelled
1D or 2D gels
Densitometer or
Photo Scanner
Normal liver
Tumor
Both
Melanie 4 (GeneBio - Windows only)
httpcaexpasyorgmelanie
ImageMaster 2D Elite (Amersham)
httpwwwimsupportcom
Phoretix 2D Advanced
httpwwwphoretixcom
PDQuest 61 (BioRad - Windows only) httpwwwproteomeworksbio-radcomhtmlpdquesthtml
Mass Spectrometry
wwwexpasych - Swiss-2DPAGE httpwwwanlgovBIOPMG - Mouse liver human breast cell lines pyrococcus Argonne Protein Mapping Group httpwwwharefieldnthamesnhsuknhliproteinindexhtml - HSC-2DPAGE Heart Science Centre Harefield Hospital httpotowustleduthcperi-gelshtm - Washington Univ Inner Ear Protein Database
httpcaexpasyorgch2d2d-indexhtml - World 2DPAGE Index of 2D gel databases
Time line ~ 4 days
bull Protein gt 250 kDa do not enter 2 SDS-PAGE properly
bull PH more than 9
bull PH less than 4
bull Hydrophobic proteins
bull Low abundant proteins
Immunoblotting Proteins from 2D gels were transferred to PVDFmembranes The membranes were blocked with 5 skimmed milk in PBS-Tween (PBS-T) for 3 h The membranes were incubated overnight at 4degC with individual sera diluted 1100 in PBS-T followed by secondary antibody (anti-human IgG conjugated with horseradish peroxidase) for 2 h with dilutions of 13000 Before and after addition of the secondary antibody membranes washed 4 times each time for 5 min in PBS-T To visualize antigenic components membranes were incubated in diaminobenzeden (DAB) and H2O2 for 15 min
List of protein spots that reacted with more than 30 of serum samples
and were identified by mass spectrometry
1Heat shock protein HSP 90-beta
2 precursor(GRP 78) 78 kDa glucose-regulated protein
3 Heat shock cognate 71 kDa protein
4 Stress-70 protein mitochondrial precursor
5 Tubulin beta-5 chain
6Tubulin alpha-6 chain and 60 kDa heat shock protein mitochondrial
precursor (Hsp60)
7 Heterogeneous nuclear ribonucleoprotein K (hnRNP K)
8 Cytokeratin 8
9 Cytokeratin 18
10 Beta-actin
11 cytokeratin 19
12 Eukaryotic initiation factor 4A-I
13 14-3-3 protein epsilon
14 14-3-3 protein zetadelta (Protein kinase Cinhibitor protein-1)
15 Translationally controlled tumor protein (TCTP)
Among these autoantigens cytokeratin 8 cytokeratin 18 Fructose-bisphosphate aldolase and enolase were reacted with more than 60 of healthy individualsrsquo serum samples These antigens were intense spots in gels
Heat Shock proteins tubulins heterogeneous nuclear ribonucleoprotein actins cytokeratins 14-3-3 protein epsilon 14-3-3 protein zetadelta eukaryotic initiation factors Rho GDP-dissociation inhibitors phosphoglycerate mutase 1 G6PD GA3PD peroxiredoxins F-acting capping protein and triosephosphate isomerase were previously reported to induce humoral arm of the immune response in cancer
Translationally controlled tumor protein and electron transfer flavoprotein alpha-subunit have not been reported to induce humoral arm of the immune response in cancer so far
In terms of biological processes the identified proteins
belong to several groups
They are mainely involved in cell motility cytoskleton
stress response apoptosis cell signaling and
metabolism
Identification of other immunogenic proteins is
underway
Appel RD et al 1993 - SWISS-2DPAGE a database of two-dimensional gel electrophoresis images Electrophoresis 14 1232-1238
Appel RD Bairoch A Sanchez JC Vargas JR Golaz O Pasquali C and Hochstrasser DF 1996 ndash Federated two-dimensional electrophoresis database a simple means of publishing two-dimensional electrophoresis data Electrophoresis 17 540-546
Bjellqvist B Ek K Righetti PG Gianazza E Gorg A Westermeier R Postel W 1982 ndash Isoelectric focusing in immobilised pH gradients principle methodology and applications JBiochemBiophysMethods 6 317-339
Brazma A et al 2001 ndash Minimum information about a microarray experiment (MIAME)-towards standards for microarray data Nat genetics 29 365-71
Hoogland C Baujard Sanchez JC Hochstrasser DF and Appel RD 1997 ndash Make2ddb a simple package to set up a two-diensional electrophoresis database for the world wide web Electrophoresis 18 2755-2758
OFarrell 1975 - High resolution two-dimensional electrophoresis of proteins JBiolChem 25 250 4007-21
Amersham Biosciences Bio-Rad
IPG strips (3 mm x 18 cm x 05 mm)
Narrow range
Medium range
Broad range
4 7
35 45 55 67
40 50 60
3 10
6 11
Load 400 ml sample per groove (no bubbles)
Place IPG strip gel facing down in groove
Add ~25 ml non conductive oil per groove
Peel off protective film from strip
Rehydrate overnight (~22 hrs) at room temperature
Program power supply
bull Number of gels (1-10)
bull Max voltage 5000 V
bull Vhold 125 V
bull Duration 24 hrs 00 min
bull Max current 80 mAstrip
bull Volt hours 80000 Vh
pI
+
+ - - - - - -
+ + + + + + + -
pH 4 pH 7
Cathode Anode
Place wet wicks (DH2O) under each end of strip
Set chiller temperature for 20degC (setting ~25)
Remove IPG strip from tray
Let oil drip off the strip
Place IPG strip gel facing up in equilibration tray
+ -
Add 10 ml equilibration buffer 1 per tray
+ -
+ -
+ -
+ -
(in chemical hood)
MW 4 7
+ -
10 DuracrylTM gel (22 cm x 23 cm x 1 mm)
bull TrisTricineSDS buffer
bull IPG strip
bull MW (gel worm)
bull Agarose overlay
bull Silicone spacer
bull Gasket
-
+
Conditions
bull Number of gel (1-10)
bull Max voltage 500 V
bull Max power 1600 mWgel
bull 4degC (setting 10)
bull ~19 hrs
10 DuracrylTM gel (22 cm x 23 cm x 1 mm)
MW 4 7
+ -
Trisacetate 25 mM Tris-base pH 83 (acetic acid)
200 mM Tris-base 200 mM Tricine 04 SDS
045 mm filtered
TrisTricine SDS
Bio-Rad
Digest to peptide fragment MS analysis
1 First dimension
isoelectric focusing
separation according to the pI
2 Second dimension
SDS electrophoresis (SDS-PAGE)
Separation according to the MW
Interested spot
Sensitivity Process
TimeSteps
Advantages
bullBio-safe Coomassie 10ng 25hr3 steps MS compatible easily visualised non
hazardous
bullCoomassie Blue R-250 40ng 25hr2 steps Oldest and least expensive method
bullSilver Stain Plus 1ng 15hr3 steps MS compatible high sensitivity low
background
bullBio-Rad silver stain 1ng 2hr7 steps High sensitivity detects some highly
glycosylated and other difficult to
stain proteins
bullSypro ruby 1ng 3hr2 steps MS compatible allows analysis in
flourescent imagers linear over 3
orders of magnitude
Coomassie staining
bull moderate sensitivity (36-47 ng)
bull non specific
bull not quantitative
Silver staining
bull sensitive (05-12 ng)
bull time consuming
bull non specific
bull negative stain some spots
Fluorescent dye (SYPRO ruby)
bull sensitive (1-2 ng)
bull specific quantitative
bull end point stain
Coomassie Silver
Coomassie Silver Stain Copper Stain
SDS-polyacrylamide gel
Electrotransfer to membrane
Phosphoimager for 32P and 35S labelled
1D or 2D gels
Fluoroimager for
SYPRO labelled
1D or 2D gels
Densitometer or
Photo Scanner
Normal liver
Tumor
Both
Melanie 4 (GeneBio - Windows only)
httpcaexpasyorgmelanie
ImageMaster 2D Elite (Amersham)
httpwwwimsupportcom
Phoretix 2D Advanced
httpwwwphoretixcom
PDQuest 61 (BioRad - Windows only) httpwwwproteomeworksbio-radcomhtmlpdquesthtml
Mass Spectrometry
wwwexpasych - Swiss-2DPAGE httpwwwanlgovBIOPMG - Mouse liver human breast cell lines pyrococcus Argonne Protein Mapping Group httpwwwharefieldnthamesnhsuknhliproteinindexhtml - HSC-2DPAGE Heart Science Centre Harefield Hospital httpotowustleduthcperi-gelshtm - Washington Univ Inner Ear Protein Database
httpcaexpasyorgch2d2d-indexhtml - World 2DPAGE Index of 2D gel databases
Time line ~ 4 days
bull Protein gt 250 kDa do not enter 2 SDS-PAGE properly
bull PH more than 9
bull PH less than 4
bull Hydrophobic proteins
bull Low abundant proteins
Immunoblotting Proteins from 2D gels were transferred to PVDFmembranes The membranes were blocked with 5 skimmed milk in PBS-Tween (PBS-T) for 3 h The membranes were incubated overnight at 4degC with individual sera diluted 1100 in PBS-T followed by secondary antibody (anti-human IgG conjugated with horseradish peroxidase) for 2 h with dilutions of 13000 Before and after addition of the secondary antibody membranes washed 4 times each time for 5 min in PBS-T To visualize antigenic components membranes were incubated in diaminobenzeden (DAB) and H2O2 for 15 min
List of protein spots that reacted with more than 30 of serum samples
and were identified by mass spectrometry
1Heat shock protein HSP 90-beta
2 precursor(GRP 78) 78 kDa glucose-regulated protein
3 Heat shock cognate 71 kDa protein
4 Stress-70 protein mitochondrial precursor
5 Tubulin beta-5 chain
6Tubulin alpha-6 chain and 60 kDa heat shock protein mitochondrial
precursor (Hsp60)
7 Heterogeneous nuclear ribonucleoprotein K (hnRNP K)
8 Cytokeratin 8
9 Cytokeratin 18
10 Beta-actin
11 cytokeratin 19
12 Eukaryotic initiation factor 4A-I
13 14-3-3 protein epsilon
14 14-3-3 protein zetadelta (Protein kinase Cinhibitor protein-1)
15 Translationally controlled tumor protein (TCTP)
Among these autoantigens cytokeratin 8 cytokeratin 18 Fructose-bisphosphate aldolase and enolase were reacted with more than 60 of healthy individualsrsquo serum samples These antigens were intense spots in gels
Heat Shock proteins tubulins heterogeneous nuclear ribonucleoprotein actins cytokeratins 14-3-3 protein epsilon 14-3-3 protein zetadelta eukaryotic initiation factors Rho GDP-dissociation inhibitors phosphoglycerate mutase 1 G6PD GA3PD peroxiredoxins F-acting capping protein and triosephosphate isomerase were previously reported to induce humoral arm of the immune response in cancer
Translationally controlled tumor protein and electron transfer flavoprotein alpha-subunit have not been reported to induce humoral arm of the immune response in cancer so far
In terms of biological processes the identified proteins
belong to several groups
They are mainely involved in cell motility cytoskleton
stress response apoptosis cell signaling and
metabolism
Identification of other immunogenic proteins is
underway
Appel RD et al 1993 - SWISS-2DPAGE a database of two-dimensional gel electrophoresis images Electrophoresis 14 1232-1238
Appel RD Bairoch A Sanchez JC Vargas JR Golaz O Pasquali C and Hochstrasser DF 1996 ndash Federated two-dimensional electrophoresis database a simple means of publishing two-dimensional electrophoresis data Electrophoresis 17 540-546
Bjellqvist B Ek K Righetti PG Gianazza E Gorg A Westermeier R Postel W 1982 ndash Isoelectric focusing in immobilised pH gradients principle methodology and applications JBiochemBiophysMethods 6 317-339
Brazma A et al 2001 ndash Minimum information about a microarray experiment (MIAME)-towards standards for microarray data Nat genetics 29 365-71
Hoogland C Baujard Sanchez JC Hochstrasser DF and Appel RD 1997 ndash Make2ddb a simple package to set up a two-diensional electrophoresis database for the world wide web Electrophoresis 18 2755-2758
OFarrell 1975 - High resolution two-dimensional electrophoresis of proteins JBiolChem 25 250 4007-21
IPG strips (3 mm x 18 cm x 05 mm)
Narrow range
Medium range
Broad range
4 7
35 45 55 67
40 50 60
3 10
6 11
Load 400 ml sample per groove (no bubbles)
Place IPG strip gel facing down in groove
Add ~25 ml non conductive oil per groove
Peel off protective film from strip
Rehydrate overnight (~22 hrs) at room temperature
Program power supply
bull Number of gels (1-10)
bull Max voltage 5000 V
bull Vhold 125 V
bull Duration 24 hrs 00 min
bull Max current 80 mAstrip
bull Volt hours 80000 Vh
pI
+
+ - - - - - -
+ + + + + + + -
pH 4 pH 7
Cathode Anode
Place wet wicks (DH2O) under each end of strip
Set chiller temperature for 20degC (setting ~25)
Remove IPG strip from tray
Let oil drip off the strip
Place IPG strip gel facing up in equilibration tray
+ -
Add 10 ml equilibration buffer 1 per tray
+ -
+ -
+ -
+ -
(in chemical hood)
MW 4 7
+ -
10 DuracrylTM gel (22 cm x 23 cm x 1 mm)
bull TrisTricineSDS buffer
bull IPG strip
bull MW (gel worm)
bull Agarose overlay
bull Silicone spacer
bull Gasket
-
+
Conditions
bull Number of gel (1-10)
bull Max voltage 500 V
bull Max power 1600 mWgel
bull 4degC (setting 10)
bull ~19 hrs
10 DuracrylTM gel (22 cm x 23 cm x 1 mm)
MW 4 7
+ -
Trisacetate 25 mM Tris-base pH 83 (acetic acid)
200 mM Tris-base 200 mM Tricine 04 SDS
045 mm filtered
TrisTricine SDS
Bio-Rad
Digest to peptide fragment MS analysis
1 First dimension
isoelectric focusing
separation according to the pI
2 Second dimension
SDS electrophoresis (SDS-PAGE)
Separation according to the MW
Interested spot
Sensitivity Process
TimeSteps
Advantages
bullBio-safe Coomassie 10ng 25hr3 steps MS compatible easily visualised non
hazardous
bullCoomassie Blue R-250 40ng 25hr2 steps Oldest and least expensive method
bullSilver Stain Plus 1ng 15hr3 steps MS compatible high sensitivity low
background
bullBio-Rad silver stain 1ng 2hr7 steps High sensitivity detects some highly
glycosylated and other difficult to
stain proteins
bullSypro ruby 1ng 3hr2 steps MS compatible allows analysis in
flourescent imagers linear over 3
orders of magnitude
Coomassie staining
bull moderate sensitivity (36-47 ng)
bull non specific
bull not quantitative
Silver staining
bull sensitive (05-12 ng)
bull time consuming
bull non specific
bull negative stain some spots
Fluorescent dye (SYPRO ruby)
bull sensitive (1-2 ng)
bull specific quantitative
bull end point stain
Coomassie Silver
Coomassie Silver Stain Copper Stain
SDS-polyacrylamide gel
Electrotransfer to membrane
Phosphoimager for 32P and 35S labelled
1D or 2D gels
Fluoroimager for
SYPRO labelled
1D or 2D gels
Densitometer or
Photo Scanner
Normal liver
Tumor
Both
Melanie 4 (GeneBio - Windows only)
httpcaexpasyorgmelanie
ImageMaster 2D Elite (Amersham)
httpwwwimsupportcom
Phoretix 2D Advanced
httpwwwphoretixcom
PDQuest 61 (BioRad - Windows only) httpwwwproteomeworksbio-radcomhtmlpdquesthtml
Mass Spectrometry
wwwexpasych - Swiss-2DPAGE httpwwwanlgovBIOPMG - Mouse liver human breast cell lines pyrococcus Argonne Protein Mapping Group httpwwwharefieldnthamesnhsuknhliproteinindexhtml - HSC-2DPAGE Heart Science Centre Harefield Hospital httpotowustleduthcperi-gelshtm - Washington Univ Inner Ear Protein Database
httpcaexpasyorgch2d2d-indexhtml - World 2DPAGE Index of 2D gel databases
Time line ~ 4 days
bull Protein gt 250 kDa do not enter 2 SDS-PAGE properly
bull PH more than 9
bull PH less than 4
bull Hydrophobic proteins
bull Low abundant proteins
Immunoblotting Proteins from 2D gels were transferred to PVDFmembranes The membranes were blocked with 5 skimmed milk in PBS-Tween (PBS-T) for 3 h The membranes were incubated overnight at 4degC with individual sera diluted 1100 in PBS-T followed by secondary antibody (anti-human IgG conjugated with horseradish peroxidase) for 2 h with dilutions of 13000 Before and after addition of the secondary antibody membranes washed 4 times each time for 5 min in PBS-T To visualize antigenic components membranes were incubated in diaminobenzeden (DAB) and H2O2 for 15 min
List of protein spots that reacted with more than 30 of serum samples
and were identified by mass spectrometry
1Heat shock protein HSP 90-beta
2 precursor(GRP 78) 78 kDa glucose-regulated protein
3 Heat shock cognate 71 kDa protein
4 Stress-70 protein mitochondrial precursor
5 Tubulin beta-5 chain
6Tubulin alpha-6 chain and 60 kDa heat shock protein mitochondrial
precursor (Hsp60)
7 Heterogeneous nuclear ribonucleoprotein K (hnRNP K)
8 Cytokeratin 8
9 Cytokeratin 18
10 Beta-actin
11 cytokeratin 19
12 Eukaryotic initiation factor 4A-I
13 14-3-3 protein epsilon
14 14-3-3 protein zetadelta (Protein kinase Cinhibitor protein-1)
15 Translationally controlled tumor protein (TCTP)
Among these autoantigens cytokeratin 8 cytokeratin 18 Fructose-bisphosphate aldolase and enolase were reacted with more than 60 of healthy individualsrsquo serum samples These antigens were intense spots in gels
Heat Shock proteins tubulins heterogeneous nuclear ribonucleoprotein actins cytokeratins 14-3-3 protein epsilon 14-3-3 protein zetadelta eukaryotic initiation factors Rho GDP-dissociation inhibitors phosphoglycerate mutase 1 G6PD GA3PD peroxiredoxins F-acting capping protein and triosephosphate isomerase were previously reported to induce humoral arm of the immune response in cancer
Translationally controlled tumor protein and electron transfer flavoprotein alpha-subunit have not been reported to induce humoral arm of the immune response in cancer so far
In terms of biological processes the identified proteins
belong to several groups
They are mainely involved in cell motility cytoskleton
stress response apoptosis cell signaling and
metabolism
Identification of other immunogenic proteins is
underway
Appel RD et al 1993 - SWISS-2DPAGE a database of two-dimensional gel electrophoresis images Electrophoresis 14 1232-1238
Appel RD Bairoch A Sanchez JC Vargas JR Golaz O Pasquali C and Hochstrasser DF 1996 ndash Federated two-dimensional electrophoresis database a simple means of publishing two-dimensional electrophoresis data Electrophoresis 17 540-546
Bjellqvist B Ek K Righetti PG Gianazza E Gorg A Westermeier R Postel W 1982 ndash Isoelectric focusing in immobilised pH gradients principle methodology and applications JBiochemBiophysMethods 6 317-339
Brazma A et al 2001 ndash Minimum information about a microarray experiment (MIAME)-towards standards for microarray data Nat genetics 29 365-71
Hoogland C Baujard Sanchez JC Hochstrasser DF and Appel RD 1997 ndash Make2ddb a simple package to set up a two-diensional electrophoresis database for the world wide web Electrophoresis 18 2755-2758
OFarrell 1975 - High resolution two-dimensional electrophoresis of proteins JBiolChem 25 250 4007-21
Load 400 ml sample per groove (no bubbles)
Place IPG strip gel facing down in groove
Add ~25 ml non conductive oil per groove
Peel off protective film from strip
Rehydrate overnight (~22 hrs) at room temperature
Program power supply
bull Number of gels (1-10)
bull Max voltage 5000 V
bull Vhold 125 V
bull Duration 24 hrs 00 min
bull Max current 80 mAstrip
bull Volt hours 80000 Vh
pI
+
+ - - - - - -
+ + + + + + + -
pH 4 pH 7
Cathode Anode
Place wet wicks (DH2O) under each end of strip
Set chiller temperature for 20degC (setting ~25)
Remove IPG strip from tray
Let oil drip off the strip
Place IPG strip gel facing up in equilibration tray
+ -
Add 10 ml equilibration buffer 1 per tray
+ -
+ -
+ -
+ -
(in chemical hood)
MW 4 7
+ -
10 DuracrylTM gel (22 cm x 23 cm x 1 mm)
bull TrisTricineSDS buffer
bull IPG strip
bull MW (gel worm)
bull Agarose overlay
bull Silicone spacer
bull Gasket
-
+
Conditions
bull Number of gel (1-10)
bull Max voltage 500 V
bull Max power 1600 mWgel
bull 4degC (setting 10)
bull ~19 hrs
10 DuracrylTM gel (22 cm x 23 cm x 1 mm)
MW 4 7
+ -
Trisacetate 25 mM Tris-base pH 83 (acetic acid)
200 mM Tris-base 200 mM Tricine 04 SDS
045 mm filtered
TrisTricine SDS
Bio-Rad
Digest to peptide fragment MS analysis
1 First dimension
isoelectric focusing
separation according to the pI
2 Second dimension
SDS electrophoresis (SDS-PAGE)
Separation according to the MW
Interested spot
Sensitivity Process
TimeSteps
Advantages
bullBio-safe Coomassie 10ng 25hr3 steps MS compatible easily visualised non
hazardous
bullCoomassie Blue R-250 40ng 25hr2 steps Oldest and least expensive method
bullSilver Stain Plus 1ng 15hr3 steps MS compatible high sensitivity low
background
bullBio-Rad silver stain 1ng 2hr7 steps High sensitivity detects some highly
glycosylated and other difficult to
stain proteins
bullSypro ruby 1ng 3hr2 steps MS compatible allows analysis in
flourescent imagers linear over 3
orders of magnitude
Coomassie staining
bull moderate sensitivity (36-47 ng)
bull non specific
bull not quantitative
Silver staining
bull sensitive (05-12 ng)
bull time consuming
bull non specific
bull negative stain some spots
Fluorescent dye (SYPRO ruby)
bull sensitive (1-2 ng)
bull specific quantitative
bull end point stain
Coomassie Silver
Coomassie Silver Stain Copper Stain
SDS-polyacrylamide gel
Electrotransfer to membrane
Phosphoimager for 32P and 35S labelled
1D or 2D gels
Fluoroimager for
SYPRO labelled
1D or 2D gels
Densitometer or
Photo Scanner
Normal liver
Tumor
Both
Melanie 4 (GeneBio - Windows only)
httpcaexpasyorgmelanie
ImageMaster 2D Elite (Amersham)
httpwwwimsupportcom
Phoretix 2D Advanced
httpwwwphoretixcom
PDQuest 61 (BioRad - Windows only) httpwwwproteomeworksbio-radcomhtmlpdquesthtml
Mass Spectrometry
wwwexpasych - Swiss-2DPAGE httpwwwanlgovBIOPMG - Mouse liver human breast cell lines pyrococcus Argonne Protein Mapping Group httpwwwharefieldnthamesnhsuknhliproteinindexhtml - HSC-2DPAGE Heart Science Centre Harefield Hospital httpotowustleduthcperi-gelshtm - Washington Univ Inner Ear Protein Database
httpcaexpasyorgch2d2d-indexhtml - World 2DPAGE Index of 2D gel databases
Time line ~ 4 days
bull Protein gt 250 kDa do not enter 2 SDS-PAGE properly
bull PH more than 9
bull PH less than 4
bull Hydrophobic proteins
bull Low abundant proteins
Immunoblotting Proteins from 2D gels were transferred to PVDFmembranes The membranes were blocked with 5 skimmed milk in PBS-Tween (PBS-T) for 3 h The membranes were incubated overnight at 4degC with individual sera diluted 1100 in PBS-T followed by secondary antibody (anti-human IgG conjugated with horseradish peroxidase) for 2 h with dilutions of 13000 Before and after addition of the secondary antibody membranes washed 4 times each time for 5 min in PBS-T To visualize antigenic components membranes were incubated in diaminobenzeden (DAB) and H2O2 for 15 min
List of protein spots that reacted with more than 30 of serum samples
and were identified by mass spectrometry
1Heat shock protein HSP 90-beta
2 precursor(GRP 78) 78 kDa glucose-regulated protein
3 Heat shock cognate 71 kDa protein
4 Stress-70 protein mitochondrial precursor
5 Tubulin beta-5 chain
6Tubulin alpha-6 chain and 60 kDa heat shock protein mitochondrial
precursor (Hsp60)
7 Heterogeneous nuclear ribonucleoprotein K (hnRNP K)
8 Cytokeratin 8
9 Cytokeratin 18
10 Beta-actin
11 cytokeratin 19
12 Eukaryotic initiation factor 4A-I
13 14-3-3 protein epsilon
14 14-3-3 protein zetadelta (Protein kinase Cinhibitor protein-1)
15 Translationally controlled tumor protein (TCTP)
Among these autoantigens cytokeratin 8 cytokeratin 18 Fructose-bisphosphate aldolase and enolase were reacted with more than 60 of healthy individualsrsquo serum samples These antigens were intense spots in gels
Heat Shock proteins tubulins heterogeneous nuclear ribonucleoprotein actins cytokeratins 14-3-3 protein epsilon 14-3-3 protein zetadelta eukaryotic initiation factors Rho GDP-dissociation inhibitors phosphoglycerate mutase 1 G6PD GA3PD peroxiredoxins F-acting capping protein and triosephosphate isomerase were previously reported to induce humoral arm of the immune response in cancer
Translationally controlled tumor protein and electron transfer flavoprotein alpha-subunit have not been reported to induce humoral arm of the immune response in cancer so far
In terms of biological processes the identified proteins
belong to several groups
They are mainely involved in cell motility cytoskleton
stress response apoptosis cell signaling and
metabolism
Identification of other immunogenic proteins is
underway
Appel RD et al 1993 - SWISS-2DPAGE a database of two-dimensional gel electrophoresis images Electrophoresis 14 1232-1238
Appel RD Bairoch A Sanchez JC Vargas JR Golaz O Pasquali C and Hochstrasser DF 1996 ndash Federated two-dimensional electrophoresis database a simple means of publishing two-dimensional electrophoresis data Electrophoresis 17 540-546
Bjellqvist B Ek K Righetti PG Gianazza E Gorg A Westermeier R Postel W 1982 ndash Isoelectric focusing in immobilised pH gradients principle methodology and applications JBiochemBiophysMethods 6 317-339
Brazma A et al 2001 ndash Minimum information about a microarray experiment (MIAME)-towards standards for microarray data Nat genetics 29 365-71
Hoogland C Baujard Sanchez JC Hochstrasser DF and Appel RD 1997 ndash Make2ddb a simple package to set up a two-diensional electrophoresis database for the world wide web Electrophoresis 18 2755-2758
OFarrell 1975 - High resolution two-dimensional electrophoresis of proteins JBiolChem 25 250 4007-21
Program power supply
bull Number of gels (1-10)
bull Max voltage 5000 V
bull Vhold 125 V
bull Duration 24 hrs 00 min
bull Max current 80 mAstrip
bull Volt hours 80000 Vh
pI
+
+ - - - - - -
+ + + + + + + -
pH 4 pH 7
Cathode Anode
Place wet wicks (DH2O) under each end of strip
Set chiller temperature for 20degC (setting ~25)
Remove IPG strip from tray
Let oil drip off the strip
Place IPG strip gel facing up in equilibration tray
+ -
Add 10 ml equilibration buffer 1 per tray
+ -
+ -
+ -
+ -
(in chemical hood)
MW 4 7
+ -
10 DuracrylTM gel (22 cm x 23 cm x 1 mm)
bull TrisTricineSDS buffer
bull IPG strip
bull MW (gel worm)
bull Agarose overlay
bull Silicone spacer
bull Gasket
-
+
Conditions
bull Number of gel (1-10)
bull Max voltage 500 V
bull Max power 1600 mWgel
bull 4degC (setting 10)
bull ~19 hrs
10 DuracrylTM gel (22 cm x 23 cm x 1 mm)
MW 4 7
+ -
Trisacetate 25 mM Tris-base pH 83 (acetic acid)
200 mM Tris-base 200 mM Tricine 04 SDS
045 mm filtered
TrisTricine SDS
Bio-Rad
Digest to peptide fragment MS analysis
1 First dimension
isoelectric focusing
separation according to the pI
2 Second dimension
SDS electrophoresis (SDS-PAGE)
Separation according to the MW
Interested spot
Sensitivity Process
TimeSteps
Advantages
bullBio-safe Coomassie 10ng 25hr3 steps MS compatible easily visualised non
hazardous
bullCoomassie Blue R-250 40ng 25hr2 steps Oldest and least expensive method
bullSilver Stain Plus 1ng 15hr3 steps MS compatible high sensitivity low
background
bullBio-Rad silver stain 1ng 2hr7 steps High sensitivity detects some highly
glycosylated and other difficult to
stain proteins
bullSypro ruby 1ng 3hr2 steps MS compatible allows analysis in
flourescent imagers linear over 3
orders of magnitude
Coomassie staining
bull moderate sensitivity (36-47 ng)
bull non specific
bull not quantitative
Silver staining
bull sensitive (05-12 ng)
bull time consuming
bull non specific
bull negative stain some spots
Fluorescent dye (SYPRO ruby)
bull sensitive (1-2 ng)
bull specific quantitative
bull end point stain
Coomassie Silver
Coomassie Silver Stain Copper Stain
SDS-polyacrylamide gel
Electrotransfer to membrane
Phosphoimager for 32P and 35S labelled
1D or 2D gels
Fluoroimager for
SYPRO labelled
1D or 2D gels
Densitometer or
Photo Scanner
Normal liver
Tumor
Both
Melanie 4 (GeneBio - Windows only)
httpcaexpasyorgmelanie
ImageMaster 2D Elite (Amersham)
httpwwwimsupportcom
Phoretix 2D Advanced
httpwwwphoretixcom
PDQuest 61 (BioRad - Windows only) httpwwwproteomeworksbio-radcomhtmlpdquesthtml
Mass Spectrometry
wwwexpasych - Swiss-2DPAGE httpwwwanlgovBIOPMG - Mouse liver human breast cell lines pyrococcus Argonne Protein Mapping Group httpwwwharefieldnthamesnhsuknhliproteinindexhtml - HSC-2DPAGE Heart Science Centre Harefield Hospital httpotowustleduthcperi-gelshtm - Washington Univ Inner Ear Protein Database
httpcaexpasyorgch2d2d-indexhtml - World 2DPAGE Index of 2D gel databases
Time line ~ 4 days
bull Protein gt 250 kDa do not enter 2 SDS-PAGE properly
bull PH more than 9
bull PH less than 4
bull Hydrophobic proteins
bull Low abundant proteins
Immunoblotting Proteins from 2D gels were transferred to PVDFmembranes The membranes were blocked with 5 skimmed milk in PBS-Tween (PBS-T) for 3 h The membranes were incubated overnight at 4degC with individual sera diluted 1100 in PBS-T followed by secondary antibody (anti-human IgG conjugated with horseradish peroxidase) for 2 h with dilutions of 13000 Before and after addition of the secondary antibody membranes washed 4 times each time for 5 min in PBS-T To visualize antigenic components membranes were incubated in diaminobenzeden (DAB) and H2O2 for 15 min
List of protein spots that reacted with more than 30 of serum samples
and were identified by mass spectrometry
1Heat shock protein HSP 90-beta
2 precursor(GRP 78) 78 kDa glucose-regulated protein
3 Heat shock cognate 71 kDa protein
4 Stress-70 protein mitochondrial precursor
5 Tubulin beta-5 chain
6Tubulin alpha-6 chain and 60 kDa heat shock protein mitochondrial
precursor (Hsp60)
7 Heterogeneous nuclear ribonucleoprotein K (hnRNP K)
8 Cytokeratin 8
9 Cytokeratin 18
10 Beta-actin
11 cytokeratin 19
12 Eukaryotic initiation factor 4A-I
13 14-3-3 protein epsilon
14 14-3-3 protein zetadelta (Protein kinase Cinhibitor protein-1)
15 Translationally controlled tumor protein (TCTP)
Among these autoantigens cytokeratin 8 cytokeratin 18 Fructose-bisphosphate aldolase and enolase were reacted with more than 60 of healthy individualsrsquo serum samples These antigens were intense spots in gels
Heat Shock proteins tubulins heterogeneous nuclear ribonucleoprotein actins cytokeratins 14-3-3 protein epsilon 14-3-3 protein zetadelta eukaryotic initiation factors Rho GDP-dissociation inhibitors phosphoglycerate mutase 1 G6PD GA3PD peroxiredoxins F-acting capping protein and triosephosphate isomerase were previously reported to induce humoral arm of the immune response in cancer
Translationally controlled tumor protein and electron transfer flavoprotein alpha-subunit have not been reported to induce humoral arm of the immune response in cancer so far
In terms of biological processes the identified proteins
belong to several groups
They are mainely involved in cell motility cytoskleton
stress response apoptosis cell signaling and
metabolism
Identification of other immunogenic proteins is
underway
Appel RD et al 1993 - SWISS-2DPAGE a database of two-dimensional gel electrophoresis images Electrophoresis 14 1232-1238
Appel RD Bairoch A Sanchez JC Vargas JR Golaz O Pasquali C and Hochstrasser DF 1996 ndash Federated two-dimensional electrophoresis database a simple means of publishing two-dimensional electrophoresis data Electrophoresis 17 540-546
Bjellqvist B Ek K Righetti PG Gianazza E Gorg A Westermeier R Postel W 1982 ndash Isoelectric focusing in immobilised pH gradients principle methodology and applications JBiochemBiophysMethods 6 317-339
Brazma A et al 2001 ndash Minimum information about a microarray experiment (MIAME)-towards standards for microarray data Nat genetics 29 365-71
Hoogland C Baujard Sanchez JC Hochstrasser DF and Appel RD 1997 ndash Make2ddb a simple package to set up a two-diensional electrophoresis database for the world wide web Electrophoresis 18 2755-2758
OFarrell 1975 - High resolution two-dimensional electrophoresis of proteins JBiolChem 25 250 4007-21
Remove IPG strip from tray
Let oil drip off the strip
Place IPG strip gel facing up in equilibration tray
+ -
Add 10 ml equilibration buffer 1 per tray
+ -
+ -
+ -
+ -
(in chemical hood)
MW 4 7
+ -
10 DuracrylTM gel (22 cm x 23 cm x 1 mm)
bull TrisTricineSDS buffer
bull IPG strip
bull MW (gel worm)
bull Agarose overlay
bull Silicone spacer
bull Gasket
-
+
Conditions
bull Number of gel (1-10)
bull Max voltage 500 V
bull Max power 1600 mWgel
bull 4degC (setting 10)
bull ~19 hrs
10 DuracrylTM gel (22 cm x 23 cm x 1 mm)
MW 4 7
+ -
Trisacetate 25 mM Tris-base pH 83 (acetic acid)
200 mM Tris-base 200 mM Tricine 04 SDS
045 mm filtered
TrisTricine SDS
Bio-Rad
Digest to peptide fragment MS analysis
1 First dimension
isoelectric focusing
separation according to the pI
2 Second dimension
SDS electrophoresis (SDS-PAGE)
Separation according to the MW
Interested spot
Sensitivity Process
TimeSteps
Advantages
bullBio-safe Coomassie 10ng 25hr3 steps MS compatible easily visualised non
hazardous
bullCoomassie Blue R-250 40ng 25hr2 steps Oldest and least expensive method
bullSilver Stain Plus 1ng 15hr3 steps MS compatible high sensitivity low
background
bullBio-Rad silver stain 1ng 2hr7 steps High sensitivity detects some highly
glycosylated and other difficult to
stain proteins
bullSypro ruby 1ng 3hr2 steps MS compatible allows analysis in
flourescent imagers linear over 3
orders of magnitude
Coomassie staining
bull moderate sensitivity (36-47 ng)
bull non specific
bull not quantitative
Silver staining
bull sensitive (05-12 ng)
bull time consuming
bull non specific
bull negative stain some spots
Fluorescent dye (SYPRO ruby)
bull sensitive (1-2 ng)
bull specific quantitative
bull end point stain
Coomassie Silver
Coomassie Silver Stain Copper Stain
SDS-polyacrylamide gel
Electrotransfer to membrane
Phosphoimager for 32P and 35S labelled
1D or 2D gels
Fluoroimager for
SYPRO labelled
1D or 2D gels
Densitometer or
Photo Scanner
Normal liver
Tumor
Both
Melanie 4 (GeneBio - Windows only)
httpcaexpasyorgmelanie
ImageMaster 2D Elite (Amersham)
httpwwwimsupportcom
Phoretix 2D Advanced
httpwwwphoretixcom
PDQuest 61 (BioRad - Windows only) httpwwwproteomeworksbio-radcomhtmlpdquesthtml
Mass Spectrometry
wwwexpasych - Swiss-2DPAGE httpwwwanlgovBIOPMG - Mouse liver human breast cell lines pyrococcus Argonne Protein Mapping Group httpwwwharefieldnthamesnhsuknhliproteinindexhtml - HSC-2DPAGE Heart Science Centre Harefield Hospital httpotowustleduthcperi-gelshtm - Washington Univ Inner Ear Protein Database
httpcaexpasyorgch2d2d-indexhtml - World 2DPAGE Index of 2D gel databases
Time line ~ 4 days
bull Protein gt 250 kDa do not enter 2 SDS-PAGE properly
bull PH more than 9
bull PH less than 4
bull Hydrophobic proteins
bull Low abundant proteins
Immunoblotting Proteins from 2D gels were transferred to PVDFmembranes The membranes were blocked with 5 skimmed milk in PBS-Tween (PBS-T) for 3 h The membranes were incubated overnight at 4degC with individual sera diluted 1100 in PBS-T followed by secondary antibody (anti-human IgG conjugated with horseradish peroxidase) for 2 h with dilutions of 13000 Before and after addition of the secondary antibody membranes washed 4 times each time for 5 min in PBS-T To visualize antigenic components membranes were incubated in diaminobenzeden (DAB) and H2O2 for 15 min
List of protein spots that reacted with more than 30 of serum samples
and were identified by mass spectrometry
1Heat shock protein HSP 90-beta
2 precursor(GRP 78) 78 kDa glucose-regulated protein
3 Heat shock cognate 71 kDa protein
4 Stress-70 protein mitochondrial precursor
5 Tubulin beta-5 chain
6Tubulin alpha-6 chain and 60 kDa heat shock protein mitochondrial
precursor (Hsp60)
7 Heterogeneous nuclear ribonucleoprotein K (hnRNP K)
8 Cytokeratin 8
9 Cytokeratin 18
10 Beta-actin
11 cytokeratin 19
12 Eukaryotic initiation factor 4A-I
13 14-3-3 protein epsilon
14 14-3-3 protein zetadelta (Protein kinase Cinhibitor protein-1)
15 Translationally controlled tumor protein (TCTP)
Among these autoantigens cytokeratin 8 cytokeratin 18 Fructose-bisphosphate aldolase and enolase were reacted with more than 60 of healthy individualsrsquo serum samples These antigens were intense spots in gels
Heat Shock proteins tubulins heterogeneous nuclear ribonucleoprotein actins cytokeratins 14-3-3 protein epsilon 14-3-3 protein zetadelta eukaryotic initiation factors Rho GDP-dissociation inhibitors phosphoglycerate mutase 1 G6PD GA3PD peroxiredoxins F-acting capping protein and triosephosphate isomerase were previously reported to induce humoral arm of the immune response in cancer
Translationally controlled tumor protein and electron transfer flavoprotein alpha-subunit have not been reported to induce humoral arm of the immune response in cancer so far
In terms of biological processes the identified proteins
belong to several groups
They are mainely involved in cell motility cytoskleton
stress response apoptosis cell signaling and
metabolism
Identification of other immunogenic proteins is
underway
Appel RD et al 1993 - SWISS-2DPAGE a database of two-dimensional gel electrophoresis images Electrophoresis 14 1232-1238
Appel RD Bairoch A Sanchez JC Vargas JR Golaz O Pasquali C and Hochstrasser DF 1996 ndash Federated two-dimensional electrophoresis database a simple means of publishing two-dimensional electrophoresis data Electrophoresis 17 540-546
Bjellqvist B Ek K Righetti PG Gianazza E Gorg A Westermeier R Postel W 1982 ndash Isoelectric focusing in immobilised pH gradients principle methodology and applications JBiochemBiophysMethods 6 317-339
Brazma A et al 2001 ndash Minimum information about a microarray experiment (MIAME)-towards standards for microarray data Nat genetics 29 365-71
Hoogland C Baujard Sanchez JC Hochstrasser DF and Appel RD 1997 ndash Make2ddb a simple package to set up a two-diensional electrophoresis database for the world wide web Electrophoresis 18 2755-2758
OFarrell 1975 - High resolution two-dimensional electrophoresis of proteins JBiolChem 25 250 4007-21
+ -
+ -
+ -
+ -
(in chemical hood)
MW 4 7
+ -
10 DuracrylTM gel (22 cm x 23 cm x 1 mm)
bull TrisTricineSDS buffer
bull IPG strip
bull MW (gel worm)
bull Agarose overlay
bull Silicone spacer
bull Gasket
-
+
Conditions
bull Number of gel (1-10)
bull Max voltage 500 V
bull Max power 1600 mWgel
bull 4degC (setting 10)
bull ~19 hrs
10 DuracrylTM gel (22 cm x 23 cm x 1 mm)
MW 4 7
+ -
Trisacetate 25 mM Tris-base pH 83 (acetic acid)
200 mM Tris-base 200 mM Tricine 04 SDS
045 mm filtered
TrisTricine SDS
Bio-Rad
Digest to peptide fragment MS analysis
1 First dimension
isoelectric focusing
separation according to the pI
2 Second dimension
SDS electrophoresis (SDS-PAGE)
Separation according to the MW
Interested spot
Sensitivity Process
TimeSteps
Advantages
bullBio-safe Coomassie 10ng 25hr3 steps MS compatible easily visualised non
hazardous
bullCoomassie Blue R-250 40ng 25hr2 steps Oldest and least expensive method
bullSilver Stain Plus 1ng 15hr3 steps MS compatible high sensitivity low
background
bullBio-Rad silver stain 1ng 2hr7 steps High sensitivity detects some highly
glycosylated and other difficult to
stain proteins
bullSypro ruby 1ng 3hr2 steps MS compatible allows analysis in
flourescent imagers linear over 3
orders of magnitude
Coomassie staining
bull moderate sensitivity (36-47 ng)
bull non specific
bull not quantitative
Silver staining
bull sensitive (05-12 ng)
bull time consuming
bull non specific
bull negative stain some spots
Fluorescent dye (SYPRO ruby)
bull sensitive (1-2 ng)
bull specific quantitative
bull end point stain
Coomassie Silver
Coomassie Silver Stain Copper Stain
SDS-polyacrylamide gel
Electrotransfer to membrane
Phosphoimager for 32P and 35S labelled
1D or 2D gels
Fluoroimager for
SYPRO labelled
1D or 2D gels
Densitometer or
Photo Scanner
Normal liver
Tumor
Both
Melanie 4 (GeneBio - Windows only)
httpcaexpasyorgmelanie
ImageMaster 2D Elite (Amersham)
httpwwwimsupportcom
Phoretix 2D Advanced
httpwwwphoretixcom
PDQuest 61 (BioRad - Windows only) httpwwwproteomeworksbio-radcomhtmlpdquesthtml
Mass Spectrometry
wwwexpasych - Swiss-2DPAGE httpwwwanlgovBIOPMG - Mouse liver human breast cell lines pyrococcus Argonne Protein Mapping Group httpwwwharefieldnthamesnhsuknhliproteinindexhtml - HSC-2DPAGE Heart Science Centre Harefield Hospital httpotowustleduthcperi-gelshtm - Washington Univ Inner Ear Protein Database
httpcaexpasyorgch2d2d-indexhtml - World 2DPAGE Index of 2D gel databases
Time line ~ 4 days
bull Protein gt 250 kDa do not enter 2 SDS-PAGE properly
bull PH more than 9
bull PH less than 4
bull Hydrophobic proteins
bull Low abundant proteins
Immunoblotting Proteins from 2D gels were transferred to PVDFmembranes The membranes were blocked with 5 skimmed milk in PBS-Tween (PBS-T) for 3 h The membranes were incubated overnight at 4degC with individual sera diluted 1100 in PBS-T followed by secondary antibody (anti-human IgG conjugated with horseradish peroxidase) for 2 h with dilutions of 13000 Before and after addition of the secondary antibody membranes washed 4 times each time for 5 min in PBS-T To visualize antigenic components membranes were incubated in diaminobenzeden (DAB) and H2O2 for 15 min
List of protein spots that reacted with more than 30 of serum samples
and were identified by mass spectrometry
1Heat shock protein HSP 90-beta
2 precursor(GRP 78) 78 kDa glucose-regulated protein
3 Heat shock cognate 71 kDa protein
4 Stress-70 protein mitochondrial precursor
5 Tubulin beta-5 chain
6Tubulin alpha-6 chain and 60 kDa heat shock protein mitochondrial
precursor (Hsp60)
7 Heterogeneous nuclear ribonucleoprotein K (hnRNP K)
8 Cytokeratin 8
9 Cytokeratin 18
10 Beta-actin
11 cytokeratin 19
12 Eukaryotic initiation factor 4A-I
13 14-3-3 protein epsilon
14 14-3-3 protein zetadelta (Protein kinase Cinhibitor protein-1)
15 Translationally controlled tumor protein (TCTP)
Among these autoantigens cytokeratin 8 cytokeratin 18 Fructose-bisphosphate aldolase and enolase were reacted with more than 60 of healthy individualsrsquo serum samples These antigens were intense spots in gels
Heat Shock proteins tubulins heterogeneous nuclear ribonucleoprotein actins cytokeratins 14-3-3 protein epsilon 14-3-3 protein zetadelta eukaryotic initiation factors Rho GDP-dissociation inhibitors phosphoglycerate mutase 1 G6PD GA3PD peroxiredoxins F-acting capping protein and triosephosphate isomerase were previously reported to induce humoral arm of the immune response in cancer
Translationally controlled tumor protein and electron transfer flavoprotein alpha-subunit have not been reported to induce humoral arm of the immune response in cancer so far
In terms of biological processes the identified proteins
belong to several groups
They are mainely involved in cell motility cytoskleton
stress response apoptosis cell signaling and
metabolism
Identification of other immunogenic proteins is
underway
Appel RD et al 1993 - SWISS-2DPAGE a database of two-dimensional gel electrophoresis images Electrophoresis 14 1232-1238
Appel RD Bairoch A Sanchez JC Vargas JR Golaz O Pasquali C and Hochstrasser DF 1996 ndash Federated two-dimensional electrophoresis database a simple means of publishing two-dimensional electrophoresis data Electrophoresis 17 540-546
Bjellqvist B Ek K Righetti PG Gianazza E Gorg A Westermeier R Postel W 1982 ndash Isoelectric focusing in immobilised pH gradients principle methodology and applications JBiochemBiophysMethods 6 317-339
Brazma A et al 2001 ndash Minimum information about a microarray experiment (MIAME)-towards standards for microarray data Nat genetics 29 365-71
Hoogland C Baujard Sanchez JC Hochstrasser DF and Appel RD 1997 ndash Make2ddb a simple package to set up a two-diensional electrophoresis database for the world wide web Electrophoresis 18 2755-2758
OFarrell 1975 - High resolution two-dimensional electrophoresis of proteins JBiolChem 25 250 4007-21
MW 4 7
+ -
10 DuracrylTM gel (22 cm x 23 cm x 1 mm)
bull TrisTricineSDS buffer
bull IPG strip
bull MW (gel worm)
bull Agarose overlay
bull Silicone spacer
bull Gasket
-
+
Conditions
bull Number of gel (1-10)
bull Max voltage 500 V
bull Max power 1600 mWgel
bull 4degC (setting 10)
bull ~19 hrs
10 DuracrylTM gel (22 cm x 23 cm x 1 mm)
MW 4 7
+ -
Trisacetate 25 mM Tris-base pH 83 (acetic acid)
200 mM Tris-base 200 mM Tricine 04 SDS
045 mm filtered
TrisTricine SDS
Bio-Rad
Digest to peptide fragment MS analysis
1 First dimension
isoelectric focusing
separation according to the pI
2 Second dimension
SDS electrophoresis (SDS-PAGE)
Separation according to the MW
Interested spot
Sensitivity Process
TimeSteps
Advantages
bullBio-safe Coomassie 10ng 25hr3 steps MS compatible easily visualised non
hazardous
bullCoomassie Blue R-250 40ng 25hr2 steps Oldest and least expensive method
bullSilver Stain Plus 1ng 15hr3 steps MS compatible high sensitivity low
background
bullBio-Rad silver stain 1ng 2hr7 steps High sensitivity detects some highly
glycosylated and other difficult to
stain proteins
bullSypro ruby 1ng 3hr2 steps MS compatible allows analysis in
flourescent imagers linear over 3
orders of magnitude
Coomassie staining
bull moderate sensitivity (36-47 ng)
bull non specific
bull not quantitative
Silver staining
bull sensitive (05-12 ng)
bull time consuming
bull non specific
bull negative stain some spots
Fluorescent dye (SYPRO ruby)
bull sensitive (1-2 ng)
bull specific quantitative
bull end point stain
Coomassie Silver
Coomassie Silver Stain Copper Stain
SDS-polyacrylamide gel
Electrotransfer to membrane
Phosphoimager for 32P and 35S labelled
1D or 2D gels
Fluoroimager for
SYPRO labelled
1D or 2D gels
Densitometer or
Photo Scanner
Normal liver
Tumor
Both
Melanie 4 (GeneBio - Windows only)
httpcaexpasyorgmelanie
ImageMaster 2D Elite (Amersham)
httpwwwimsupportcom
Phoretix 2D Advanced
httpwwwphoretixcom
PDQuest 61 (BioRad - Windows only) httpwwwproteomeworksbio-radcomhtmlpdquesthtml
Mass Spectrometry
wwwexpasych - Swiss-2DPAGE httpwwwanlgovBIOPMG - Mouse liver human breast cell lines pyrococcus Argonne Protein Mapping Group httpwwwharefieldnthamesnhsuknhliproteinindexhtml - HSC-2DPAGE Heart Science Centre Harefield Hospital httpotowustleduthcperi-gelshtm - Washington Univ Inner Ear Protein Database
httpcaexpasyorgch2d2d-indexhtml - World 2DPAGE Index of 2D gel databases
Time line ~ 4 days
bull Protein gt 250 kDa do not enter 2 SDS-PAGE properly
bull PH more than 9
bull PH less than 4
bull Hydrophobic proteins
bull Low abundant proteins
Immunoblotting Proteins from 2D gels were transferred to PVDFmembranes The membranes were blocked with 5 skimmed milk in PBS-Tween (PBS-T) for 3 h The membranes were incubated overnight at 4degC with individual sera diluted 1100 in PBS-T followed by secondary antibody (anti-human IgG conjugated with horseradish peroxidase) for 2 h with dilutions of 13000 Before and after addition of the secondary antibody membranes washed 4 times each time for 5 min in PBS-T To visualize antigenic components membranes were incubated in diaminobenzeden (DAB) and H2O2 for 15 min
List of protein spots that reacted with more than 30 of serum samples
and were identified by mass spectrometry
1Heat shock protein HSP 90-beta
2 precursor(GRP 78) 78 kDa glucose-regulated protein
3 Heat shock cognate 71 kDa protein
4 Stress-70 protein mitochondrial precursor
5 Tubulin beta-5 chain
6Tubulin alpha-6 chain and 60 kDa heat shock protein mitochondrial
precursor (Hsp60)
7 Heterogeneous nuclear ribonucleoprotein K (hnRNP K)
8 Cytokeratin 8
9 Cytokeratin 18
10 Beta-actin
11 cytokeratin 19
12 Eukaryotic initiation factor 4A-I
13 14-3-3 protein epsilon
14 14-3-3 protein zetadelta (Protein kinase Cinhibitor protein-1)
15 Translationally controlled tumor protein (TCTP)
Among these autoantigens cytokeratin 8 cytokeratin 18 Fructose-bisphosphate aldolase and enolase were reacted with more than 60 of healthy individualsrsquo serum samples These antigens were intense spots in gels
Heat Shock proteins tubulins heterogeneous nuclear ribonucleoprotein actins cytokeratins 14-3-3 protein epsilon 14-3-3 protein zetadelta eukaryotic initiation factors Rho GDP-dissociation inhibitors phosphoglycerate mutase 1 G6PD GA3PD peroxiredoxins F-acting capping protein and triosephosphate isomerase were previously reported to induce humoral arm of the immune response in cancer
Translationally controlled tumor protein and electron transfer flavoprotein alpha-subunit have not been reported to induce humoral arm of the immune response in cancer so far
In terms of biological processes the identified proteins
belong to several groups
They are mainely involved in cell motility cytoskleton
stress response apoptosis cell signaling and
metabolism
Identification of other immunogenic proteins is
underway
Appel RD et al 1993 - SWISS-2DPAGE a database of two-dimensional gel electrophoresis images Electrophoresis 14 1232-1238
Appel RD Bairoch A Sanchez JC Vargas JR Golaz O Pasquali C and Hochstrasser DF 1996 ndash Federated two-dimensional electrophoresis database a simple means of publishing two-dimensional electrophoresis data Electrophoresis 17 540-546
Bjellqvist B Ek K Righetti PG Gianazza E Gorg A Westermeier R Postel W 1982 ndash Isoelectric focusing in immobilised pH gradients principle methodology and applications JBiochemBiophysMethods 6 317-339
Brazma A et al 2001 ndash Minimum information about a microarray experiment (MIAME)-towards standards for microarray data Nat genetics 29 365-71
Hoogland C Baujard Sanchez JC Hochstrasser DF and Appel RD 1997 ndash Make2ddb a simple package to set up a two-diensional electrophoresis database for the world wide web Electrophoresis 18 2755-2758
OFarrell 1975 - High resolution two-dimensional electrophoresis of proteins JBiolChem 25 250 4007-21
-
+
Conditions
bull Number of gel (1-10)
bull Max voltage 500 V
bull Max power 1600 mWgel
bull 4degC (setting 10)
bull ~19 hrs
10 DuracrylTM gel (22 cm x 23 cm x 1 mm)
MW 4 7
+ -
Trisacetate 25 mM Tris-base pH 83 (acetic acid)
200 mM Tris-base 200 mM Tricine 04 SDS
045 mm filtered
TrisTricine SDS
Bio-Rad
Digest to peptide fragment MS analysis
1 First dimension
isoelectric focusing
separation according to the pI
2 Second dimension
SDS electrophoresis (SDS-PAGE)
Separation according to the MW
Interested spot
Sensitivity Process
TimeSteps
Advantages
bullBio-safe Coomassie 10ng 25hr3 steps MS compatible easily visualised non
hazardous
bullCoomassie Blue R-250 40ng 25hr2 steps Oldest and least expensive method
bullSilver Stain Plus 1ng 15hr3 steps MS compatible high sensitivity low
background
bullBio-Rad silver stain 1ng 2hr7 steps High sensitivity detects some highly
glycosylated and other difficult to
stain proteins
bullSypro ruby 1ng 3hr2 steps MS compatible allows analysis in
flourescent imagers linear over 3
orders of magnitude
Coomassie staining
bull moderate sensitivity (36-47 ng)
bull non specific
bull not quantitative
Silver staining
bull sensitive (05-12 ng)
bull time consuming
bull non specific
bull negative stain some spots
Fluorescent dye (SYPRO ruby)
bull sensitive (1-2 ng)
bull specific quantitative
bull end point stain
Coomassie Silver
Coomassie Silver Stain Copper Stain
SDS-polyacrylamide gel
Electrotransfer to membrane
Phosphoimager for 32P and 35S labelled
1D or 2D gels
Fluoroimager for
SYPRO labelled
1D or 2D gels
Densitometer or
Photo Scanner
Normal liver
Tumor
Both
Melanie 4 (GeneBio - Windows only)
httpcaexpasyorgmelanie
ImageMaster 2D Elite (Amersham)
httpwwwimsupportcom
Phoretix 2D Advanced
httpwwwphoretixcom
PDQuest 61 (BioRad - Windows only) httpwwwproteomeworksbio-radcomhtmlpdquesthtml
Mass Spectrometry
wwwexpasych - Swiss-2DPAGE httpwwwanlgovBIOPMG - Mouse liver human breast cell lines pyrococcus Argonne Protein Mapping Group httpwwwharefieldnthamesnhsuknhliproteinindexhtml - HSC-2DPAGE Heart Science Centre Harefield Hospital httpotowustleduthcperi-gelshtm - Washington Univ Inner Ear Protein Database
httpcaexpasyorgch2d2d-indexhtml - World 2DPAGE Index of 2D gel databases
Time line ~ 4 days
bull Protein gt 250 kDa do not enter 2 SDS-PAGE properly
bull PH more than 9
bull PH less than 4
bull Hydrophobic proteins
bull Low abundant proteins
Immunoblotting Proteins from 2D gels were transferred to PVDFmembranes The membranes were blocked with 5 skimmed milk in PBS-Tween (PBS-T) for 3 h The membranes were incubated overnight at 4degC with individual sera diluted 1100 in PBS-T followed by secondary antibody (anti-human IgG conjugated with horseradish peroxidase) for 2 h with dilutions of 13000 Before and after addition of the secondary antibody membranes washed 4 times each time for 5 min in PBS-T To visualize antigenic components membranes were incubated in diaminobenzeden (DAB) and H2O2 for 15 min
List of protein spots that reacted with more than 30 of serum samples
and were identified by mass spectrometry
1Heat shock protein HSP 90-beta
2 precursor(GRP 78) 78 kDa glucose-regulated protein
3 Heat shock cognate 71 kDa protein
4 Stress-70 protein mitochondrial precursor
5 Tubulin beta-5 chain
6Tubulin alpha-6 chain and 60 kDa heat shock protein mitochondrial
precursor (Hsp60)
7 Heterogeneous nuclear ribonucleoprotein K (hnRNP K)
8 Cytokeratin 8
9 Cytokeratin 18
10 Beta-actin
11 cytokeratin 19
12 Eukaryotic initiation factor 4A-I
13 14-3-3 protein epsilon
14 14-3-3 protein zetadelta (Protein kinase Cinhibitor protein-1)
15 Translationally controlled tumor protein (TCTP)
Among these autoantigens cytokeratin 8 cytokeratin 18 Fructose-bisphosphate aldolase and enolase were reacted with more than 60 of healthy individualsrsquo serum samples These antigens were intense spots in gels
Heat Shock proteins tubulins heterogeneous nuclear ribonucleoprotein actins cytokeratins 14-3-3 protein epsilon 14-3-3 protein zetadelta eukaryotic initiation factors Rho GDP-dissociation inhibitors phosphoglycerate mutase 1 G6PD GA3PD peroxiredoxins F-acting capping protein and triosephosphate isomerase were previously reported to induce humoral arm of the immune response in cancer
Translationally controlled tumor protein and electron transfer flavoprotein alpha-subunit have not been reported to induce humoral arm of the immune response in cancer so far
In terms of biological processes the identified proteins
belong to several groups
They are mainely involved in cell motility cytoskleton
stress response apoptosis cell signaling and
metabolism
Identification of other immunogenic proteins is
underway
Appel RD et al 1993 - SWISS-2DPAGE a database of two-dimensional gel electrophoresis images Electrophoresis 14 1232-1238
Appel RD Bairoch A Sanchez JC Vargas JR Golaz O Pasquali C and Hochstrasser DF 1996 ndash Federated two-dimensional electrophoresis database a simple means of publishing two-dimensional electrophoresis data Electrophoresis 17 540-546
Bjellqvist B Ek K Righetti PG Gianazza E Gorg A Westermeier R Postel W 1982 ndash Isoelectric focusing in immobilised pH gradients principle methodology and applications JBiochemBiophysMethods 6 317-339
Brazma A et al 2001 ndash Minimum information about a microarray experiment (MIAME)-towards standards for microarray data Nat genetics 29 365-71
Hoogland C Baujard Sanchez JC Hochstrasser DF and Appel RD 1997 ndash Make2ddb a simple package to set up a two-diensional electrophoresis database for the world wide web Electrophoresis 18 2755-2758
OFarrell 1975 - High resolution two-dimensional electrophoresis of proteins JBiolChem 25 250 4007-21
Bio-Rad
Digest to peptide fragment MS analysis
1 First dimension
isoelectric focusing
separation according to the pI
2 Second dimension
SDS electrophoresis (SDS-PAGE)
Separation according to the MW
Interested spot
Sensitivity Process
TimeSteps
Advantages
bullBio-safe Coomassie 10ng 25hr3 steps MS compatible easily visualised non
hazardous
bullCoomassie Blue R-250 40ng 25hr2 steps Oldest and least expensive method
bullSilver Stain Plus 1ng 15hr3 steps MS compatible high sensitivity low
background
bullBio-Rad silver stain 1ng 2hr7 steps High sensitivity detects some highly
glycosylated and other difficult to
stain proteins
bullSypro ruby 1ng 3hr2 steps MS compatible allows analysis in
flourescent imagers linear over 3
orders of magnitude
Coomassie staining
bull moderate sensitivity (36-47 ng)
bull non specific
bull not quantitative
Silver staining
bull sensitive (05-12 ng)
bull time consuming
bull non specific
bull negative stain some spots
Fluorescent dye (SYPRO ruby)
bull sensitive (1-2 ng)
bull specific quantitative
bull end point stain
Coomassie Silver
Coomassie Silver Stain Copper Stain
SDS-polyacrylamide gel
Electrotransfer to membrane
Phosphoimager for 32P and 35S labelled
1D or 2D gels
Fluoroimager for
SYPRO labelled
1D or 2D gels
Densitometer or
Photo Scanner
Normal liver
Tumor
Both
Melanie 4 (GeneBio - Windows only)
httpcaexpasyorgmelanie
ImageMaster 2D Elite (Amersham)
httpwwwimsupportcom
Phoretix 2D Advanced
httpwwwphoretixcom
PDQuest 61 (BioRad - Windows only) httpwwwproteomeworksbio-radcomhtmlpdquesthtml
Mass Spectrometry
wwwexpasych - Swiss-2DPAGE httpwwwanlgovBIOPMG - Mouse liver human breast cell lines pyrococcus Argonne Protein Mapping Group httpwwwharefieldnthamesnhsuknhliproteinindexhtml - HSC-2DPAGE Heart Science Centre Harefield Hospital httpotowustleduthcperi-gelshtm - Washington Univ Inner Ear Protein Database
httpcaexpasyorgch2d2d-indexhtml - World 2DPAGE Index of 2D gel databases
Time line ~ 4 days
bull Protein gt 250 kDa do not enter 2 SDS-PAGE properly
bull PH more than 9
bull PH less than 4
bull Hydrophobic proteins
bull Low abundant proteins
Immunoblotting Proteins from 2D gels were transferred to PVDFmembranes The membranes were blocked with 5 skimmed milk in PBS-Tween (PBS-T) for 3 h The membranes were incubated overnight at 4degC with individual sera diluted 1100 in PBS-T followed by secondary antibody (anti-human IgG conjugated with horseradish peroxidase) for 2 h with dilutions of 13000 Before and after addition of the secondary antibody membranes washed 4 times each time for 5 min in PBS-T To visualize antigenic components membranes were incubated in diaminobenzeden (DAB) and H2O2 for 15 min
List of protein spots that reacted with more than 30 of serum samples
and were identified by mass spectrometry
1Heat shock protein HSP 90-beta
2 precursor(GRP 78) 78 kDa glucose-regulated protein
3 Heat shock cognate 71 kDa protein
4 Stress-70 protein mitochondrial precursor
5 Tubulin beta-5 chain
6Tubulin alpha-6 chain and 60 kDa heat shock protein mitochondrial
precursor (Hsp60)
7 Heterogeneous nuclear ribonucleoprotein K (hnRNP K)
8 Cytokeratin 8
9 Cytokeratin 18
10 Beta-actin
11 cytokeratin 19
12 Eukaryotic initiation factor 4A-I
13 14-3-3 protein epsilon
14 14-3-3 protein zetadelta (Protein kinase Cinhibitor protein-1)
15 Translationally controlled tumor protein (TCTP)
Among these autoantigens cytokeratin 8 cytokeratin 18 Fructose-bisphosphate aldolase and enolase were reacted with more than 60 of healthy individualsrsquo serum samples These antigens were intense spots in gels
Heat Shock proteins tubulins heterogeneous nuclear ribonucleoprotein actins cytokeratins 14-3-3 protein epsilon 14-3-3 protein zetadelta eukaryotic initiation factors Rho GDP-dissociation inhibitors phosphoglycerate mutase 1 G6PD GA3PD peroxiredoxins F-acting capping protein and triosephosphate isomerase were previously reported to induce humoral arm of the immune response in cancer
Translationally controlled tumor protein and electron transfer flavoprotein alpha-subunit have not been reported to induce humoral arm of the immune response in cancer so far
In terms of biological processes the identified proteins
belong to several groups
They are mainely involved in cell motility cytoskleton
stress response apoptosis cell signaling and
metabolism
Identification of other immunogenic proteins is
underway
Appel RD et al 1993 - SWISS-2DPAGE a database of two-dimensional gel electrophoresis images Electrophoresis 14 1232-1238
Appel RD Bairoch A Sanchez JC Vargas JR Golaz O Pasquali C and Hochstrasser DF 1996 ndash Federated two-dimensional electrophoresis database a simple means of publishing two-dimensional electrophoresis data Electrophoresis 17 540-546
Bjellqvist B Ek K Righetti PG Gianazza E Gorg A Westermeier R Postel W 1982 ndash Isoelectric focusing in immobilised pH gradients principle methodology and applications JBiochemBiophysMethods 6 317-339
Brazma A et al 2001 ndash Minimum information about a microarray experiment (MIAME)-towards standards for microarray data Nat genetics 29 365-71
Hoogland C Baujard Sanchez JC Hochstrasser DF and Appel RD 1997 ndash Make2ddb a simple package to set up a two-diensional electrophoresis database for the world wide web Electrophoresis 18 2755-2758
OFarrell 1975 - High resolution two-dimensional electrophoresis of proteins JBiolChem 25 250 4007-21
Digest to peptide fragment MS analysis
1 First dimension
isoelectric focusing
separation according to the pI
2 Second dimension
SDS electrophoresis (SDS-PAGE)
Separation according to the MW
Interested spot
Sensitivity Process
TimeSteps
Advantages
bullBio-safe Coomassie 10ng 25hr3 steps MS compatible easily visualised non
hazardous
bullCoomassie Blue R-250 40ng 25hr2 steps Oldest and least expensive method
bullSilver Stain Plus 1ng 15hr3 steps MS compatible high sensitivity low
background
bullBio-Rad silver stain 1ng 2hr7 steps High sensitivity detects some highly
glycosylated and other difficult to
stain proteins
bullSypro ruby 1ng 3hr2 steps MS compatible allows analysis in
flourescent imagers linear over 3
orders of magnitude
Coomassie staining
bull moderate sensitivity (36-47 ng)
bull non specific
bull not quantitative
Silver staining
bull sensitive (05-12 ng)
bull time consuming
bull non specific
bull negative stain some spots
Fluorescent dye (SYPRO ruby)
bull sensitive (1-2 ng)
bull specific quantitative
bull end point stain
Coomassie Silver
Coomassie Silver Stain Copper Stain
SDS-polyacrylamide gel
Electrotransfer to membrane
Phosphoimager for 32P and 35S labelled
1D or 2D gels
Fluoroimager for
SYPRO labelled
1D or 2D gels
Densitometer or
Photo Scanner
Normal liver
Tumor
Both
Melanie 4 (GeneBio - Windows only)
httpcaexpasyorgmelanie
ImageMaster 2D Elite (Amersham)
httpwwwimsupportcom
Phoretix 2D Advanced
httpwwwphoretixcom
PDQuest 61 (BioRad - Windows only) httpwwwproteomeworksbio-radcomhtmlpdquesthtml
Mass Spectrometry
wwwexpasych - Swiss-2DPAGE httpwwwanlgovBIOPMG - Mouse liver human breast cell lines pyrococcus Argonne Protein Mapping Group httpwwwharefieldnthamesnhsuknhliproteinindexhtml - HSC-2DPAGE Heart Science Centre Harefield Hospital httpotowustleduthcperi-gelshtm - Washington Univ Inner Ear Protein Database
httpcaexpasyorgch2d2d-indexhtml - World 2DPAGE Index of 2D gel databases
Time line ~ 4 days
bull Protein gt 250 kDa do not enter 2 SDS-PAGE properly
bull PH more than 9
bull PH less than 4
bull Hydrophobic proteins
bull Low abundant proteins
Immunoblotting Proteins from 2D gels were transferred to PVDFmembranes The membranes were blocked with 5 skimmed milk in PBS-Tween (PBS-T) for 3 h The membranes were incubated overnight at 4degC with individual sera diluted 1100 in PBS-T followed by secondary antibody (anti-human IgG conjugated with horseradish peroxidase) for 2 h with dilutions of 13000 Before and after addition of the secondary antibody membranes washed 4 times each time for 5 min in PBS-T To visualize antigenic components membranes were incubated in diaminobenzeden (DAB) and H2O2 for 15 min
List of protein spots that reacted with more than 30 of serum samples
and were identified by mass spectrometry
1Heat shock protein HSP 90-beta
2 precursor(GRP 78) 78 kDa glucose-regulated protein
3 Heat shock cognate 71 kDa protein
4 Stress-70 protein mitochondrial precursor
5 Tubulin beta-5 chain
6Tubulin alpha-6 chain and 60 kDa heat shock protein mitochondrial
precursor (Hsp60)
7 Heterogeneous nuclear ribonucleoprotein K (hnRNP K)
8 Cytokeratin 8
9 Cytokeratin 18
10 Beta-actin
11 cytokeratin 19
12 Eukaryotic initiation factor 4A-I
13 14-3-3 protein epsilon
14 14-3-3 protein zetadelta (Protein kinase Cinhibitor protein-1)
15 Translationally controlled tumor protein (TCTP)
Among these autoantigens cytokeratin 8 cytokeratin 18 Fructose-bisphosphate aldolase and enolase were reacted with more than 60 of healthy individualsrsquo serum samples These antigens were intense spots in gels
Heat Shock proteins tubulins heterogeneous nuclear ribonucleoprotein actins cytokeratins 14-3-3 protein epsilon 14-3-3 protein zetadelta eukaryotic initiation factors Rho GDP-dissociation inhibitors phosphoglycerate mutase 1 G6PD GA3PD peroxiredoxins F-acting capping protein and triosephosphate isomerase were previously reported to induce humoral arm of the immune response in cancer
Translationally controlled tumor protein and electron transfer flavoprotein alpha-subunit have not been reported to induce humoral arm of the immune response in cancer so far
In terms of biological processes the identified proteins
belong to several groups
They are mainely involved in cell motility cytoskleton
stress response apoptosis cell signaling and
metabolism
Identification of other immunogenic proteins is
underway
Appel RD et al 1993 - SWISS-2DPAGE a database of two-dimensional gel electrophoresis images Electrophoresis 14 1232-1238
Appel RD Bairoch A Sanchez JC Vargas JR Golaz O Pasquali C and Hochstrasser DF 1996 ndash Federated two-dimensional electrophoresis database a simple means of publishing two-dimensional electrophoresis data Electrophoresis 17 540-546
Bjellqvist B Ek K Righetti PG Gianazza E Gorg A Westermeier R Postel W 1982 ndash Isoelectric focusing in immobilised pH gradients principle methodology and applications JBiochemBiophysMethods 6 317-339
Brazma A et al 2001 ndash Minimum information about a microarray experiment (MIAME)-towards standards for microarray data Nat genetics 29 365-71
Hoogland C Baujard Sanchez JC Hochstrasser DF and Appel RD 1997 ndash Make2ddb a simple package to set up a two-diensional electrophoresis database for the world wide web Electrophoresis 18 2755-2758
OFarrell 1975 - High resolution two-dimensional electrophoresis of proteins JBiolChem 25 250 4007-21
Sensitivity Process
TimeSteps
Advantages
bullBio-safe Coomassie 10ng 25hr3 steps MS compatible easily visualised non
hazardous
bullCoomassie Blue R-250 40ng 25hr2 steps Oldest and least expensive method
bullSilver Stain Plus 1ng 15hr3 steps MS compatible high sensitivity low
background
bullBio-Rad silver stain 1ng 2hr7 steps High sensitivity detects some highly
glycosylated and other difficult to
stain proteins
bullSypro ruby 1ng 3hr2 steps MS compatible allows analysis in
flourescent imagers linear over 3
orders of magnitude
Coomassie staining
bull moderate sensitivity (36-47 ng)
bull non specific
bull not quantitative
Silver staining
bull sensitive (05-12 ng)
bull time consuming
bull non specific
bull negative stain some spots
Fluorescent dye (SYPRO ruby)
bull sensitive (1-2 ng)
bull specific quantitative
bull end point stain
Coomassie Silver
Coomassie Silver Stain Copper Stain
SDS-polyacrylamide gel
Electrotransfer to membrane
Phosphoimager for 32P and 35S labelled
1D or 2D gels
Fluoroimager for
SYPRO labelled
1D or 2D gels
Densitometer or
Photo Scanner
Normal liver
Tumor
Both
Melanie 4 (GeneBio - Windows only)
httpcaexpasyorgmelanie
ImageMaster 2D Elite (Amersham)
httpwwwimsupportcom
Phoretix 2D Advanced
httpwwwphoretixcom
PDQuest 61 (BioRad - Windows only) httpwwwproteomeworksbio-radcomhtmlpdquesthtml
Mass Spectrometry
wwwexpasych - Swiss-2DPAGE httpwwwanlgovBIOPMG - Mouse liver human breast cell lines pyrococcus Argonne Protein Mapping Group httpwwwharefieldnthamesnhsuknhliproteinindexhtml - HSC-2DPAGE Heart Science Centre Harefield Hospital httpotowustleduthcperi-gelshtm - Washington Univ Inner Ear Protein Database
httpcaexpasyorgch2d2d-indexhtml - World 2DPAGE Index of 2D gel databases
Time line ~ 4 days
bull Protein gt 250 kDa do not enter 2 SDS-PAGE properly
bull PH more than 9
bull PH less than 4
bull Hydrophobic proteins
bull Low abundant proteins
Immunoblotting Proteins from 2D gels were transferred to PVDFmembranes The membranes were blocked with 5 skimmed milk in PBS-Tween (PBS-T) for 3 h The membranes were incubated overnight at 4degC with individual sera diluted 1100 in PBS-T followed by secondary antibody (anti-human IgG conjugated with horseradish peroxidase) for 2 h with dilutions of 13000 Before and after addition of the secondary antibody membranes washed 4 times each time for 5 min in PBS-T To visualize antigenic components membranes were incubated in diaminobenzeden (DAB) and H2O2 for 15 min
List of protein spots that reacted with more than 30 of serum samples
and were identified by mass spectrometry
1Heat shock protein HSP 90-beta
2 precursor(GRP 78) 78 kDa glucose-regulated protein
3 Heat shock cognate 71 kDa protein
4 Stress-70 protein mitochondrial precursor
5 Tubulin beta-5 chain
6Tubulin alpha-6 chain and 60 kDa heat shock protein mitochondrial
precursor (Hsp60)
7 Heterogeneous nuclear ribonucleoprotein K (hnRNP K)
8 Cytokeratin 8
9 Cytokeratin 18
10 Beta-actin
11 cytokeratin 19
12 Eukaryotic initiation factor 4A-I
13 14-3-3 protein epsilon
14 14-3-3 protein zetadelta (Protein kinase Cinhibitor protein-1)
15 Translationally controlled tumor protein (TCTP)
Among these autoantigens cytokeratin 8 cytokeratin 18 Fructose-bisphosphate aldolase and enolase were reacted with more than 60 of healthy individualsrsquo serum samples These antigens were intense spots in gels
Heat Shock proteins tubulins heterogeneous nuclear ribonucleoprotein actins cytokeratins 14-3-3 protein epsilon 14-3-3 protein zetadelta eukaryotic initiation factors Rho GDP-dissociation inhibitors phosphoglycerate mutase 1 G6PD GA3PD peroxiredoxins F-acting capping protein and triosephosphate isomerase were previously reported to induce humoral arm of the immune response in cancer
Translationally controlled tumor protein and electron transfer flavoprotein alpha-subunit have not been reported to induce humoral arm of the immune response in cancer so far
In terms of biological processes the identified proteins
belong to several groups
They are mainely involved in cell motility cytoskleton
stress response apoptosis cell signaling and
metabolism
Identification of other immunogenic proteins is
underway
Appel RD et al 1993 - SWISS-2DPAGE a database of two-dimensional gel electrophoresis images Electrophoresis 14 1232-1238
Appel RD Bairoch A Sanchez JC Vargas JR Golaz O Pasquali C and Hochstrasser DF 1996 ndash Federated two-dimensional electrophoresis database a simple means of publishing two-dimensional electrophoresis data Electrophoresis 17 540-546
Bjellqvist B Ek K Righetti PG Gianazza E Gorg A Westermeier R Postel W 1982 ndash Isoelectric focusing in immobilised pH gradients principle methodology and applications JBiochemBiophysMethods 6 317-339
Brazma A et al 2001 ndash Minimum information about a microarray experiment (MIAME)-towards standards for microarray data Nat genetics 29 365-71
Hoogland C Baujard Sanchez JC Hochstrasser DF and Appel RD 1997 ndash Make2ddb a simple package to set up a two-diensional electrophoresis database for the world wide web Electrophoresis 18 2755-2758
OFarrell 1975 - High resolution two-dimensional electrophoresis of proteins JBiolChem 25 250 4007-21
Coomassie staining
bull moderate sensitivity (36-47 ng)
bull non specific
bull not quantitative
Silver staining
bull sensitive (05-12 ng)
bull time consuming
bull non specific
bull negative stain some spots
Fluorescent dye (SYPRO ruby)
bull sensitive (1-2 ng)
bull specific quantitative
bull end point stain
Coomassie Silver
Coomassie Silver Stain Copper Stain
SDS-polyacrylamide gel
Electrotransfer to membrane
Phosphoimager for 32P and 35S labelled
1D or 2D gels
Fluoroimager for
SYPRO labelled
1D or 2D gels
Densitometer or
Photo Scanner
Normal liver
Tumor
Both
Melanie 4 (GeneBio - Windows only)
httpcaexpasyorgmelanie
ImageMaster 2D Elite (Amersham)
httpwwwimsupportcom
Phoretix 2D Advanced
httpwwwphoretixcom
PDQuest 61 (BioRad - Windows only) httpwwwproteomeworksbio-radcomhtmlpdquesthtml
Mass Spectrometry
wwwexpasych - Swiss-2DPAGE httpwwwanlgovBIOPMG - Mouse liver human breast cell lines pyrococcus Argonne Protein Mapping Group httpwwwharefieldnthamesnhsuknhliproteinindexhtml - HSC-2DPAGE Heart Science Centre Harefield Hospital httpotowustleduthcperi-gelshtm - Washington Univ Inner Ear Protein Database
httpcaexpasyorgch2d2d-indexhtml - World 2DPAGE Index of 2D gel databases
Time line ~ 4 days
bull Protein gt 250 kDa do not enter 2 SDS-PAGE properly
bull PH more than 9
bull PH less than 4
bull Hydrophobic proteins
bull Low abundant proteins
Immunoblotting Proteins from 2D gels were transferred to PVDFmembranes The membranes were blocked with 5 skimmed milk in PBS-Tween (PBS-T) for 3 h The membranes were incubated overnight at 4degC with individual sera diluted 1100 in PBS-T followed by secondary antibody (anti-human IgG conjugated with horseradish peroxidase) for 2 h with dilutions of 13000 Before and after addition of the secondary antibody membranes washed 4 times each time for 5 min in PBS-T To visualize antigenic components membranes were incubated in diaminobenzeden (DAB) and H2O2 for 15 min
List of protein spots that reacted with more than 30 of serum samples
and were identified by mass spectrometry
1Heat shock protein HSP 90-beta
2 precursor(GRP 78) 78 kDa glucose-regulated protein
3 Heat shock cognate 71 kDa protein
4 Stress-70 protein mitochondrial precursor
5 Tubulin beta-5 chain
6Tubulin alpha-6 chain and 60 kDa heat shock protein mitochondrial
precursor (Hsp60)
7 Heterogeneous nuclear ribonucleoprotein K (hnRNP K)
8 Cytokeratin 8
9 Cytokeratin 18
10 Beta-actin
11 cytokeratin 19
12 Eukaryotic initiation factor 4A-I
13 14-3-3 protein epsilon
14 14-3-3 protein zetadelta (Protein kinase Cinhibitor protein-1)
15 Translationally controlled tumor protein (TCTP)
Among these autoantigens cytokeratin 8 cytokeratin 18 Fructose-bisphosphate aldolase and enolase were reacted with more than 60 of healthy individualsrsquo serum samples These antigens were intense spots in gels
Heat Shock proteins tubulins heterogeneous nuclear ribonucleoprotein actins cytokeratins 14-3-3 protein epsilon 14-3-3 protein zetadelta eukaryotic initiation factors Rho GDP-dissociation inhibitors phosphoglycerate mutase 1 G6PD GA3PD peroxiredoxins F-acting capping protein and triosephosphate isomerase were previously reported to induce humoral arm of the immune response in cancer
Translationally controlled tumor protein and electron transfer flavoprotein alpha-subunit have not been reported to induce humoral arm of the immune response in cancer so far
In terms of biological processes the identified proteins
belong to several groups
They are mainely involved in cell motility cytoskleton
stress response apoptosis cell signaling and
metabolism
Identification of other immunogenic proteins is
underway
Appel RD et al 1993 - SWISS-2DPAGE a database of two-dimensional gel electrophoresis images Electrophoresis 14 1232-1238
Appel RD Bairoch A Sanchez JC Vargas JR Golaz O Pasquali C and Hochstrasser DF 1996 ndash Federated two-dimensional electrophoresis database a simple means of publishing two-dimensional electrophoresis data Electrophoresis 17 540-546
Bjellqvist B Ek K Righetti PG Gianazza E Gorg A Westermeier R Postel W 1982 ndash Isoelectric focusing in immobilised pH gradients principle methodology and applications JBiochemBiophysMethods 6 317-339
Brazma A et al 2001 ndash Minimum information about a microarray experiment (MIAME)-towards standards for microarray data Nat genetics 29 365-71
Hoogland C Baujard Sanchez JC Hochstrasser DF and Appel RD 1997 ndash Make2ddb a simple package to set up a two-diensional electrophoresis database for the world wide web Electrophoresis 18 2755-2758
OFarrell 1975 - High resolution two-dimensional electrophoresis of proteins JBiolChem 25 250 4007-21
Coomassie Silver
Coomassie Silver Stain Copper Stain
SDS-polyacrylamide gel
Electrotransfer to membrane
Phosphoimager for 32P and 35S labelled
1D or 2D gels
Fluoroimager for
SYPRO labelled
1D or 2D gels
Densitometer or
Photo Scanner
Normal liver
Tumor
Both
Melanie 4 (GeneBio - Windows only)
httpcaexpasyorgmelanie
ImageMaster 2D Elite (Amersham)
httpwwwimsupportcom
Phoretix 2D Advanced
httpwwwphoretixcom
PDQuest 61 (BioRad - Windows only) httpwwwproteomeworksbio-radcomhtmlpdquesthtml
Mass Spectrometry
wwwexpasych - Swiss-2DPAGE httpwwwanlgovBIOPMG - Mouse liver human breast cell lines pyrococcus Argonne Protein Mapping Group httpwwwharefieldnthamesnhsuknhliproteinindexhtml - HSC-2DPAGE Heart Science Centre Harefield Hospital httpotowustleduthcperi-gelshtm - Washington Univ Inner Ear Protein Database
httpcaexpasyorgch2d2d-indexhtml - World 2DPAGE Index of 2D gel databases
Time line ~ 4 days
bull Protein gt 250 kDa do not enter 2 SDS-PAGE properly
bull PH more than 9
bull PH less than 4
bull Hydrophobic proteins
bull Low abundant proteins
Immunoblotting Proteins from 2D gels were transferred to PVDFmembranes The membranes were blocked with 5 skimmed milk in PBS-Tween (PBS-T) for 3 h The membranes were incubated overnight at 4degC with individual sera diluted 1100 in PBS-T followed by secondary antibody (anti-human IgG conjugated with horseradish peroxidase) for 2 h with dilutions of 13000 Before and after addition of the secondary antibody membranes washed 4 times each time for 5 min in PBS-T To visualize antigenic components membranes were incubated in diaminobenzeden (DAB) and H2O2 for 15 min
List of protein spots that reacted with more than 30 of serum samples
and were identified by mass spectrometry
1Heat shock protein HSP 90-beta
2 precursor(GRP 78) 78 kDa glucose-regulated protein
3 Heat shock cognate 71 kDa protein
4 Stress-70 protein mitochondrial precursor
5 Tubulin beta-5 chain
6Tubulin alpha-6 chain and 60 kDa heat shock protein mitochondrial
precursor (Hsp60)
7 Heterogeneous nuclear ribonucleoprotein K (hnRNP K)
8 Cytokeratin 8
9 Cytokeratin 18
10 Beta-actin
11 cytokeratin 19
12 Eukaryotic initiation factor 4A-I
13 14-3-3 protein epsilon
14 14-3-3 protein zetadelta (Protein kinase Cinhibitor protein-1)
15 Translationally controlled tumor protein (TCTP)
Among these autoantigens cytokeratin 8 cytokeratin 18 Fructose-bisphosphate aldolase and enolase were reacted with more than 60 of healthy individualsrsquo serum samples These antigens were intense spots in gels
Heat Shock proteins tubulins heterogeneous nuclear ribonucleoprotein actins cytokeratins 14-3-3 protein epsilon 14-3-3 protein zetadelta eukaryotic initiation factors Rho GDP-dissociation inhibitors phosphoglycerate mutase 1 G6PD GA3PD peroxiredoxins F-acting capping protein and triosephosphate isomerase were previously reported to induce humoral arm of the immune response in cancer
Translationally controlled tumor protein and electron transfer flavoprotein alpha-subunit have not been reported to induce humoral arm of the immune response in cancer so far
In terms of biological processes the identified proteins
belong to several groups
They are mainely involved in cell motility cytoskleton
stress response apoptosis cell signaling and
metabolism
Identification of other immunogenic proteins is
underway
Appel RD et al 1993 - SWISS-2DPAGE a database of two-dimensional gel electrophoresis images Electrophoresis 14 1232-1238
Appel RD Bairoch A Sanchez JC Vargas JR Golaz O Pasquali C and Hochstrasser DF 1996 ndash Federated two-dimensional electrophoresis database a simple means of publishing two-dimensional electrophoresis data Electrophoresis 17 540-546
Bjellqvist B Ek K Righetti PG Gianazza E Gorg A Westermeier R Postel W 1982 ndash Isoelectric focusing in immobilised pH gradients principle methodology and applications JBiochemBiophysMethods 6 317-339
Brazma A et al 2001 ndash Minimum information about a microarray experiment (MIAME)-towards standards for microarray data Nat genetics 29 365-71
Hoogland C Baujard Sanchez JC Hochstrasser DF and Appel RD 1997 ndash Make2ddb a simple package to set up a two-diensional electrophoresis database for the world wide web Electrophoresis 18 2755-2758
OFarrell 1975 - High resolution two-dimensional electrophoresis of proteins JBiolChem 25 250 4007-21
Coomassie Silver Stain Copper Stain
SDS-polyacrylamide gel
Electrotransfer to membrane
Phosphoimager for 32P and 35S labelled
1D or 2D gels
Fluoroimager for
SYPRO labelled
1D or 2D gels
Densitometer or
Photo Scanner
Normal liver
Tumor
Both
Melanie 4 (GeneBio - Windows only)
httpcaexpasyorgmelanie
ImageMaster 2D Elite (Amersham)
httpwwwimsupportcom
Phoretix 2D Advanced
httpwwwphoretixcom
PDQuest 61 (BioRad - Windows only) httpwwwproteomeworksbio-radcomhtmlpdquesthtml
Mass Spectrometry
wwwexpasych - Swiss-2DPAGE httpwwwanlgovBIOPMG - Mouse liver human breast cell lines pyrococcus Argonne Protein Mapping Group httpwwwharefieldnthamesnhsuknhliproteinindexhtml - HSC-2DPAGE Heart Science Centre Harefield Hospital httpotowustleduthcperi-gelshtm - Washington Univ Inner Ear Protein Database
httpcaexpasyorgch2d2d-indexhtml - World 2DPAGE Index of 2D gel databases
Time line ~ 4 days
bull Protein gt 250 kDa do not enter 2 SDS-PAGE properly
bull PH more than 9
bull PH less than 4
bull Hydrophobic proteins
bull Low abundant proteins
Immunoblotting Proteins from 2D gels were transferred to PVDFmembranes The membranes were blocked with 5 skimmed milk in PBS-Tween (PBS-T) for 3 h The membranes were incubated overnight at 4degC with individual sera diluted 1100 in PBS-T followed by secondary antibody (anti-human IgG conjugated with horseradish peroxidase) for 2 h with dilutions of 13000 Before and after addition of the secondary antibody membranes washed 4 times each time for 5 min in PBS-T To visualize antigenic components membranes were incubated in diaminobenzeden (DAB) and H2O2 for 15 min
List of protein spots that reacted with more than 30 of serum samples
and were identified by mass spectrometry
1Heat shock protein HSP 90-beta
2 precursor(GRP 78) 78 kDa glucose-regulated protein
3 Heat shock cognate 71 kDa protein
4 Stress-70 protein mitochondrial precursor
5 Tubulin beta-5 chain
6Tubulin alpha-6 chain and 60 kDa heat shock protein mitochondrial
precursor (Hsp60)
7 Heterogeneous nuclear ribonucleoprotein K (hnRNP K)
8 Cytokeratin 8
9 Cytokeratin 18
10 Beta-actin
11 cytokeratin 19
12 Eukaryotic initiation factor 4A-I
13 14-3-3 protein epsilon
14 14-3-3 protein zetadelta (Protein kinase Cinhibitor protein-1)
15 Translationally controlled tumor protein (TCTP)
Among these autoantigens cytokeratin 8 cytokeratin 18 Fructose-bisphosphate aldolase and enolase were reacted with more than 60 of healthy individualsrsquo serum samples These antigens were intense spots in gels
Heat Shock proteins tubulins heterogeneous nuclear ribonucleoprotein actins cytokeratins 14-3-3 protein epsilon 14-3-3 protein zetadelta eukaryotic initiation factors Rho GDP-dissociation inhibitors phosphoglycerate mutase 1 G6PD GA3PD peroxiredoxins F-acting capping protein and triosephosphate isomerase were previously reported to induce humoral arm of the immune response in cancer
Translationally controlled tumor protein and electron transfer flavoprotein alpha-subunit have not been reported to induce humoral arm of the immune response in cancer so far
In terms of biological processes the identified proteins
belong to several groups
They are mainely involved in cell motility cytoskleton
stress response apoptosis cell signaling and
metabolism
Identification of other immunogenic proteins is
underway
Appel RD et al 1993 - SWISS-2DPAGE a database of two-dimensional gel electrophoresis images Electrophoresis 14 1232-1238
Appel RD Bairoch A Sanchez JC Vargas JR Golaz O Pasquali C and Hochstrasser DF 1996 ndash Federated two-dimensional electrophoresis database a simple means of publishing two-dimensional electrophoresis data Electrophoresis 17 540-546
Bjellqvist B Ek K Righetti PG Gianazza E Gorg A Westermeier R Postel W 1982 ndash Isoelectric focusing in immobilised pH gradients principle methodology and applications JBiochemBiophysMethods 6 317-339
Brazma A et al 2001 ndash Minimum information about a microarray experiment (MIAME)-towards standards for microarray data Nat genetics 29 365-71
Hoogland C Baujard Sanchez JC Hochstrasser DF and Appel RD 1997 ndash Make2ddb a simple package to set up a two-diensional electrophoresis database for the world wide web Electrophoresis 18 2755-2758
OFarrell 1975 - High resolution two-dimensional electrophoresis of proteins JBiolChem 25 250 4007-21
SDS-polyacrylamide gel
Electrotransfer to membrane
Phosphoimager for 32P and 35S labelled
1D or 2D gels
Fluoroimager for
SYPRO labelled
1D or 2D gels
Densitometer or
Photo Scanner
Normal liver
Tumor
Both
Melanie 4 (GeneBio - Windows only)
httpcaexpasyorgmelanie
ImageMaster 2D Elite (Amersham)
httpwwwimsupportcom
Phoretix 2D Advanced
httpwwwphoretixcom
PDQuest 61 (BioRad - Windows only) httpwwwproteomeworksbio-radcomhtmlpdquesthtml
Mass Spectrometry
wwwexpasych - Swiss-2DPAGE httpwwwanlgovBIOPMG - Mouse liver human breast cell lines pyrococcus Argonne Protein Mapping Group httpwwwharefieldnthamesnhsuknhliproteinindexhtml - HSC-2DPAGE Heart Science Centre Harefield Hospital httpotowustleduthcperi-gelshtm - Washington Univ Inner Ear Protein Database
httpcaexpasyorgch2d2d-indexhtml - World 2DPAGE Index of 2D gel databases
Time line ~ 4 days
bull Protein gt 250 kDa do not enter 2 SDS-PAGE properly
bull PH more than 9
bull PH less than 4
bull Hydrophobic proteins
bull Low abundant proteins
Immunoblotting Proteins from 2D gels were transferred to PVDFmembranes The membranes were blocked with 5 skimmed milk in PBS-Tween (PBS-T) for 3 h The membranes were incubated overnight at 4degC with individual sera diluted 1100 in PBS-T followed by secondary antibody (anti-human IgG conjugated with horseradish peroxidase) for 2 h with dilutions of 13000 Before and after addition of the secondary antibody membranes washed 4 times each time for 5 min in PBS-T To visualize antigenic components membranes were incubated in diaminobenzeden (DAB) and H2O2 for 15 min
List of protein spots that reacted with more than 30 of serum samples
and were identified by mass spectrometry
1Heat shock protein HSP 90-beta
2 precursor(GRP 78) 78 kDa glucose-regulated protein
3 Heat shock cognate 71 kDa protein
4 Stress-70 protein mitochondrial precursor
5 Tubulin beta-5 chain
6Tubulin alpha-6 chain and 60 kDa heat shock protein mitochondrial
precursor (Hsp60)
7 Heterogeneous nuclear ribonucleoprotein K (hnRNP K)
8 Cytokeratin 8
9 Cytokeratin 18
10 Beta-actin
11 cytokeratin 19
12 Eukaryotic initiation factor 4A-I
13 14-3-3 protein epsilon
14 14-3-3 protein zetadelta (Protein kinase Cinhibitor protein-1)
15 Translationally controlled tumor protein (TCTP)
Among these autoantigens cytokeratin 8 cytokeratin 18 Fructose-bisphosphate aldolase and enolase were reacted with more than 60 of healthy individualsrsquo serum samples These antigens were intense spots in gels
Heat Shock proteins tubulins heterogeneous nuclear ribonucleoprotein actins cytokeratins 14-3-3 protein epsilon 14-3-3 protein zetadelta eukaryotic initiation factors Rho GDP-dissociation inhibitors phosphoglycerate mutase 1 G6PD GA3PD peroxiredoxins F-acting capping protein and triosephosphate isomerase were previously reported to induce humoral arm of the immune response in cancer
Translationally controlled tumor protein and electron transfer flavoprotein alpha-subunit have not been reported to induce humoral arm of the immune response in cancer so far
In terms of biological processes the identified proteins
belong to several groups
They are mainely involved in cell motility cytoskleton
stress response apoptosis cell signaling and
metabolism
Identification of other immunogenic proteins is
underway
Appel RD et al 1993 - SWISS-2DPAGE a database of two-dimensional gel electrophoresis images Electrophoresis 14 1232-1238
Appel RD Bairoch A Sanchez JC Vargas JR Golaz O Pasquali C and Hochstrasser DF 1996 ndash Federated two-dimensional electrophoresis database a simple means of publishing two-dimensional electrophoresis data Electrophoresis 17 540-546
Bjellqvist B Ek K Righetti PG Gianazza E Gorg A Westermeier R Postel W 1982 ndash Isoelectric focusing in immobilised pH gradients principle methodology and applications JBiochemBiophysMethods 6 317-339
Brazma A et al 2001 ndash Minimum information about a microarray experiment (MIAME)-towards standards for microarray data Nat genetics 29 365-71
Hoogland C Baujard Sanchez JC Hochstrasser DF and Appel RD 1997 ndash Make2ddb a simple package to set up a two-diensional electrophoresis database for the world wide web Electrophoresis 18 2755-2758
OFarrell 1975 - High resolution two-dimensional electrophoresis of proteins JBiolChem 25 250 4007-21
Phosphoimager for 32P and 35S labelled
1D or 2D gels
Fluoroimager for
SYPRO labelled
1D or 2D gels
Densitometer or
Photo Scanner
Normal liver
Tumor
Both
Melanie 4 (GeneBio - Windows only)
httpcaexpasyorgmelanie
ImageMaster 2D Elite (Amersham)
httpwwwimsupportcom
Phoretix 2D Advanced
httpwwwphoretixcom
PDQuest 61 (BioRad - Windows only) httpwwwproteomeworksbio-radcomhtmlpdquesthtml
Mass Spectrometry
wwwexpasych - Swiss-2DPAGE httpwwwanlgovBIOPMG - Mouse liver human breast cell lines pyrococcus Argonne Protein Mapping Group httpwwwharefieldnthamesnhsuknhliproteinindexhtml - HSC-2DPAGE Heart Science Centre Harefield Hospital httpotowustleduthcperi-gelshtm - Washington Univ Inner Ear Protein Database
httpcaexpasyorgch2d2d-indexhtml - World 2DPAGE Index of 2D gel databases
Time line ~ 4 days
bull Protein gt 250 kDa do not enter 2 SDS-PAGE properly
bull PH more than 9
bull PH less than 4
bull Hydrophobic proteins
bull Low abundant proteins
Immunoblotting Proteins from 2D gels were transferred to PVDFmembranes The membranes were blocked with 5 skimmed milk in PBS-Tween (PBS-T) for 3 h The membranes were incubated overnight at 4degC with individual sera diluted 1100 in PBS-T followed by secondary antibody (anti-human IgG conjugated with horseradish peroxidase) for 2 h with dilutions of 13000 Before and after addition of the secondary antibody membranes washed 4 times each time for 5 min in PBS-T To visualize antigenic components membranes were incubated in diaminobenzeden (DAB) and H2O2 for 15 min
List of protein spots that reacted with more than 30 of serum samples
and were identified by mass spectrometry
1Heat shock protein HSP 90-beta
2 precursor(GRP 78) 78 kDa glucose-regulated protein
3 Heat shock cognate 71 kDa protein
4 Stress-70 protein mitochondrial precursor
5 Tubulin beta-5 chain
6Tubulin alpha-6 chain and 60 kDa heat shock protein mitochondrial
precursor (Hsp60)
7 Heterogeneous nuclear ribonucleoprotein K (hnRNP K)
8 Cytokeratin 8
9 Cytokeratin 18
10 Beta-actin
11 cytokeratin 19
12 Eukaryotic initiation factor 4A-I
13 14-3-3 protein epsilon
14 14-3-3 protein zetadelta (Protein kinase Cinhibitor protein-1)
15 Translationally controlled tumor protein (TCTP)
Among these autoantigens cytokeratin 8 cytokeratin 18 Fructose-bisphosphate aldolase and enolase were reacted with more than 60 of healthy individualsrsquo serum samples These antigens were intense spots in gels
Heat Shock proteins tubulins heterogeneous nuclear ribonucleoprotein actins cytokeratins 14-3-3 protein epsilon 14-3-3 protein zetadelta eukaryotic initiation factors Rho GDP-dissociation inhibitors phosphoglycerate mutase 1 G6PD GA3PD peroxiredoxins F-acting capping protein and triosephosphate isomerase were previously reported to induce humoral arm of the immune response in cancer
Translationally controlled tumor protein and electron transfer flavoprotein alpha-subunit have not been reported to induce humoral arm of the immune response in cancer so far
In terms of biological processes the identified proteins
belong to several groups
They are mainely involved in cell motility cytoskleton
stress response apoptosis cell signaling and
metabolism
Identification of other immunogenic proteins is
underway
Appel RD et al 1993 - SWISS-2DPAGE a database of two-dimensional gel electrophoresis images Electrophoresis 14 1232-1238
Appel RD Bairoch A Sanchez JC Vargas JR Golaz O Pasquali C and Hochstrasser DF 1996 ndash Federated two-dimensional electrophoresis database a simple means of publishing two-dimensional electrophoresis data Electrophoresis 17 540-546
Bjellqvist B Ek K Righetti PG Gianazza E Gorg A Westermeier R Postel W 1982 ndash Isoelectric focusing in immobilised pH gradients principle methodology and applications JBiochemBiophysMethods 6 317-339
Brazma A et al 2001 ndash Minimum information about a microarray experiment (MIAME)-towards standards for microarray data Nat genetics 29 365-71
Hoogland C Baujard Sanchez JC Hochstrasser DF and Appel RD 1997 ndash Make2ddb a simple package to set up a two-diensional electrophoresis database for the world wide web Electrophoresis 18 2755-2758
OFarrell 1975 - High resolution two-dimensional electrophoresis of proteins JBiolChem 25 250 4007-21
Normal liver
Tumor
Both
Melanie 4 (GeneBio - Windows only)
httpcaexpasyorgmelanie
ImageMaster 2D Elite (Amersham)
httpwwwimsupportcom
Phoretix 2D Advanced
httpwwwphoretixcom
PDQuest 61 (BioRad - Windows only) httpwwwproteomeworksbio-radcomhtmlpdquesthtml
Mass Spectrometry
wwwexpasych - Swiss-2DPAGE httpwwwanlgovBIOPMG - Mouse liver human breast cell lines pyrococcus Argonne Protein Mapping Group httpwwwharefieldnthamesnhsuknhliproteinindexhtml - HSC-2DPAGE Heart Science Centre Harefield Hospital httpotowustleduthcperi-gelshtm - Washington Univ Inner Ear Protein Database
httpcaexpasyorgch2d2d-indexhtml - World 2DPAGE Index of 2D gel databases
Time line ~ 4 days
bull Protein gt 250 kDa do not enter 2 SDS-PAGE properly
bull PH more than 9
bull PH less than 4
bull Hydrophobic proteins
bull Low abundant proteins
Immunoblotting Proteins from 2D gels were transferred to PVDFmembranes The membranes were blocked with 5 skimmed milk in PBS-Tween (PBS-T) for 3 h The membranes were incubated overnight at 4degC with individual sera diluted 1100 in PBS-T followed by secondary antibody (anti-human IgG conjugated with horseradish peroxidase) for 2 h with dilutions of 13000 Before and after addition of the secondary antibody membranes washed 4 times each time for 5 min in PBS-T To visualize antigenic components membranes were incubated in diaminobenzeden (DAB) and H2O2 for 15 min
List of protein spots that reacted with more than 30 of serum samples
and were identified by mass spectrometry
1Heat shock protein HSP 90-beta
2 precursor(GRP 78) 78 kDa glucose-regulated protein
3 Heat shock cognate 71 kDa protein
4 Stress-70 protein mitochondrial precursor
5 Tubulin beta-5 chain
6Tubulin alpha-6 chain and 60 kDa heat shock protein mitochondrial
precursor (Hsp60)
7 Heterogeneous nuclear ribonucleoprotein K (hnRNP K)
8 Cytokeratin 8
9 Cytokeratin 18
10 Beta-actin
11 cytokeratin 19
12 Eukaryotic initiation factor 4A-I
13 14-3-3 protein epsilon
14 14-3-3 protein zetadelta (Protein kinase Cinhibitor protein-1)
15 Translationally controlled tumor protein (TCTP)
Among these autoantigens cytokeratin 8 cytokeratin 18 Fructose-bisphosphate aldolase and enolase were reacted with more than 60 of healthy individualsrsquo serum samples These antigens were intense spots in gels
Heat Shock proteins tubulins heterogeneous nuclear ribonucleoprotein actins cytokeratins 14-3-3 protein epsilon 14-3-3 protein zetadelta eukaryotic initiation factors Rho GDP-dissociation inhibitors phosphoglycerate mutase 1 G6PD GA3PD peroxiredoxins F-acting capping protein and triosephosphate isomerase were previously reported to induce humoral arm of the immune response in cancer
Translationally controlled tumor protein and electron transfer flavoprotein alpha-subunit have not been reported to induce humoral arm of the immune response in cancer so far
In terms of biological processes the identified proteins
belong to several groups
They are mainely involved in cell motility cytoskleton
stress response apoptosis cell signaling and
metabolism
Identification of other immunogenic proteins is
underway
Appel RD et al 1993 - SWISS-2DPAGE a database of two-dimensional gel electrophoresis images Electrophoresis 14 1232-1238
Appel RD Bairoch A Sanchez JC Vargas JR Golaz O Pasquali C and Hochstrasser DF 1996 ndash Federated two-dimensional electrophoresis database a simple means of publishing two-dimensional electrophoresis data Electrophoresis 17 540-546
Bjellqvist B Ek K Righetti PG Gianazza E Gorg A Westermeier R Postel W 1982 ndash Isoelectric focusing in immobilised pH gradients principle methodology and applications JBiochemBiophysMethods 6 317-339
Brazma A et al 2001 ndash Minimum information about a microarray experiment (MIAME)-towards standards for microarray data Nat genetics 29 365-71
Hoogland C Baujard Sanchez JC Hochstrasser DF and Appel RD 1997 ndash Make2ddb a simple package to set up a two-diensional electrophoresis database for the world wide web Electrophoresis 18 2755-2758
OFarrell 1975 - High resolution two-dimensional electrophoresis of proteins JBiolChem 25 250 4007-21
Melanie 4 (GeneBio - Windows only)
httpcaexpasyorgmelanie
ImageMaster 2D Elite (Amersham)
httpwwwimsupportcom
Phoretix 2D Advanced
httpwwwphoretixcom
PDQuest 61 (BioRad - Windows only) httpwwwproteomeworksbio-radcomhtmlpdquesthtml
Mass Spectrometry
wwwexpasych - Swiss-2DPAGE httpwwwanlgovBIOPMG - Mouse liver human breast cell lines pyrococcus Argonne Protein Mapping Group httpwwwharefieldnthamesnhsuknhliproteinindexhtml - HSC-2DPAGE Heart Science Centre Harefield Hospital httpotowustleduthcperi-gelshtm - Washington Univ Inner Ear Protein Database
httpcaexpasyorgch2d2d-indexhtml - World 2DPAGE Index of 2D gel databases
Time line ~ 4 days
bull Protein gt 250 kDa do not enter 2 SDS-PAGE properly
bull PH more than 9
bull PH less than 4
bull Hydrophobic proteins
bull Low abundant proteins
Immunoblotting Proteins from 2D gels were transferred to PVDFmembranes The membranes were blocked with 5 skimmed milk in PBS-Tween (PBS-T) for 3 h The membranes were incubated overnight at 4degC with individual sera diluted 1100 in PBS-T followed by secondary antibody (anti-human IgG conjugated with horseradish peroxidase) for 2 h with dilutions of 13000 Before and after addition of the secondary antibody membranes washed 4 times each time for 5 min in PBS-T To visualize antigenic components membranes were incubated in diaminobenzeden (DAB) and H2O2 for 15 min
List of protein spots that reacted with more than 30 of serum samples
and were identified by mass spectrometry
1Heat shock protein HSP 90-beta
2 precursor(GRP 78) 78 kDa glucose-regulated protein
3 Heat shock cognate 71 kDa protein
4 Stress-70 protein mitochondrial precursor
5 Tubulin beta-5 chain
6Tubulin alpha-6 chain and 60 kDa heat shock protein mitochondrial
precursor (Hsp60)
7 Heterogeneous nuclear ribonucleoprotein K (hnRNP K)
8 Cytokeratin 8
9 Cytokeratin 18
10 Beta-actin
11 cytokeratin 19
12 Eukaryotic initiation factor 4A-I
13 14-3-3 protein epsilon
14 14-3-3 protein zetadelta (Protein kinase Cinhibitor protein-1)
15 Translationally controlled tumor protein (TCTP)
Among these autoantigens cytokeratin 8 cytokeratin 18 Fructose-bisphosphate aldolase and enolase were reacted with more than 60 of healthy individualsrsquo serum samples These antigens were intense spots in gels
Heat Shock proteins tubulins heterogeneous nuclear ribonucleoprotein actins cytokeratins 14-3-3 protein epsilon 14-3-3 protein zetadelta eukaryotic initiation factors Rho GDP-dissociation inhibitors phosphoglycerate mutase 1 G6PD GA3PD peroxiredoxins F-acting capping protein and triosephosphate isomerase were previously reported to induce humoral arm of the immune response in cancer
Translationally controlled tumor protein and electron transfer flavoprotein alpha-subunit have not been reported to induce humoral arm of the immune response in cancer so far
In terms of biological processes the identified proteins
belong to several groups
They are mainely involved in cell motility cytoskleton
stress response apoptosis cell signaling and
metabolism
Identification of other immunogenic proteins is
underway
Appel RD et al 1993 - SWISS-2DPAGE a database of two-dimensional gel electrophoresis images Electrophoresis 14 1232-1238
Appel RD Bairoch A Sanchez JC Vargas JR Golaz O Pasquali C and Hochstrasser DF 1996 ndash Federated two-dimensional electrophoresis database a simple means of publishing two-dimensional electrophoresis data Electrophoresis 17 540-546
Bjellqvist B Ek K Righetti PG Gianazza E Gorg A Westermeier R Postel W 1982 ndash Isoelectric focusing in immobilised pH gradients principle methodology and applications JBiochemBiophysMethods 6 317-339
Brazma A et al 2001 ndash Minimum information about a microarray experiment (MIAME)-towards standards for microarray data Nat genetics 29 365-71
Hoogland C Baujard Sanchez JC Hochstrasser DF and Appel RD 1997 ndash Make2ddb a simple package to set up a two-diensional electrophoresis database for the world wide web Electrophoresis 18 2755-2758
OFarrell 1975 - High resolution two-dimensional electrophoresis of proteins JBiolChem 25 250 4007-21
Mass Spectrometry
wwwexpasych - Swiss-2DPAGE httpwwwanlgovBIOPMG - Mouse liver human breast cell lines pyrococcus Argonne Protein Mapping Group httpwwwharefieldnthamesnhsuknhliproteinindexhtml - HSC-2DPAGE Heart Science Centre Harefield Hospital httpotowustleduthcperi-gelshtm - Washington Univ Inner Ear Protein Database
httpcaexpasyorgch2d2d-indexhtml - World 2DPAGE Index of 2D gel databases
Time line ~ 4 days
bull Protein gt 250 kDa do not enter 2 SDS-PAGE properly
bull PH more than 9
bull PH less than 4
bull Hydrophobic proteins
bull Low abundant proteins
Immunoblotting Proteins from 2D gels were transferred to PVDFmembranes The membranes were blocked with 5 skimmed milk in PBS-Tween (PBS-T) for 3 h The membranes were incubated overnight at 4degC with individual sera diluted 1100 in PBS-T followed by secondary antibody (anti-human IgG conjugated with horseradish peroxidase) for 2 h with dilutions of 13000 Before and after addition of the secondary antibody membranes washed 4 times each time for 5 min in PBS-T To visualize antigenic components membranes were incubated in diaminobenzeden (DAB) and H2O2 for 15 min
List of protein spots that reacted with more than 30 of serum samples
and were identified by mass spectrometry
1Heat shock protein HSP 90-beta
2 precursor(GRP 78) 78 kDa glucose-regulated protein
3 Heat shock cognate 71 kDa protein
4 Stress-70 protein mitochondrial precursor
5 Tubulin beta-5 chain
6Tubulin alpha-6 chain and 60 kDa heat shock protein mitochondrial
precursor (Hsp60)
7 Heterogeneous nuclear ribonucleoprotein K (hnRNP K)
8 Cytokeratin 8
9 Cytokeratin 18
10 Beta-actin
11 cytokeratin 19
12 Eukaryotic initiation factor 4A-I
13 14-3-3 protein epsilon
14 14-3-3 protein zetadelta (Protein kinase Cinhibitor protein-1)
15 Translationally controlled tumor protein (TCTP)
Among these autoantigens cytokeratin 8 cytokeratin 18 Fructose-bisphosphate aldolase and enolase were reacted with more than 60 of healthy individualsrsquo serum samples These antigens were intense spots in gels
Heat Shock proteins tubulins heterogeneous nuclear ribonucleoprotein actins cytokeratins 14-3-3 protein epsilon 14-3-3 protein zetadelta eukaryotic initiation factors Rho GDP-dissociation inhibitors phosphoglycerate mutase 1 G6PD GA3PD peroxiredoxins F-acting capping protein and triosephosphate isomerase were previously reported to induce humoral arm of the immune response in cancer
Translationally controlled tumor protein and electron transfer flavoprotein alpha-subunit have not been reported to induce humoral arm of the immune response in cancer so far
In terms of biological processes the identified proteins
belong to several groups
They are mainely involved in cell motility cytoskleton
stress response apoptosis cell signaling and
metabolism
Identification of other immunogenic proteins is
underway
Appel RD et al 1993 - SWISS-2DPAGE a database of two-dimensional gel electrophoresis images Electrophoresis 14 1232-1238
Appel RD Bairoch A Sanchez JC Vargas JR Golaz O Pasquali C and Hochstrasser DF 1996 ndash Federated two-dimensional electrophoresis database a simple means of publishing two-dimensional electrophoresis data Electrophoresis 17 540-546
Bjellqvist B Ek K Righetti PG Gianazza E Gorg A Westermeier R Postel W 1982 ndash Isoelectric focusing in immobilised pH gradients principle methodology and applications JBiochemBiophysMethods 6 317-339
Brazma A et al 2001 ndash Minimum information about a microarray experiment (MIAME)-towards standards for microarray data Nat genetics 29 365-71
Hoogland C Baujard Sanchez JC Hochstrasser DF and Appel RD 1997 ndash Make2ddb a simple package to set up a two-diensional electrophoresis database for the world wide web Electrophoresis 18 2755-2758
OFarrell 1975 - High resolution two-dimensional electrophoresis of proteins JBiolChem 25 250 4007-21
wwwexpasych - Swiss-2DPAGE httpwwwanlgovBIOPMG - Mouse liver human breast cell lines pyrococcus Argonne Protein Mapping Group httpwwwharefieldnthamesnhsuknhliproteinindexhtml - HSC-2DPAGE Heart Science Centre Harefield Hospital httpotowustleduthcperi-gelshtm - Washington Univ Inner Ear Protein Database
httpcaexpasyorgch2d2d-indexhtml - World 2DPAGE Index of 2D gel databases
Time line ~ 4 days
bull Protein gt 250 kDa do not enter 2 SDS-PAGE properly
bull PH more than 9
bull PH less than 4
bull Hydrophobic proteins
bull Low abundant proteins
Immunoblotting Proteins from 2D gels were transferred to PVDFmembranes The membranes were blocked with 5 skimmed milk in PBS-Tween (PBS-T) for 3 h The membranes were incubated overnight at 4degC with individual sera diluted 1100 in PBS-T followed by secondary antibody (anti-human IgG conjugated with horseradish peroxidase) for 2 h with dilutions of 13000 Before and after addition of the secondary antibody membranes washed 4 times each time for 5 min in PBS-T To visualize antigenic components membranes were incubated in diaminobenzeden (DAB) and H2O2 for 15 min
List of protein spots that reacted with more than 30 of serum samples
and were identified by mass spectrometry
1Heat shock protein HSP 90-beta
2 precursor(GRP 78) 78 kDa glucose-regulated protein
3 Heat shock cognate 71 kDa protein
4 Stress-70 protein mitochondrial precursor
5 Tubulin beta-5 chain
6Tubulin alpha-6 chain and 60 kDa heat shock protein mitochondrial
precursor (Hsp60)
7 Heterogeneous nuclear ribonucleoprotein K (hnRNP K)
8 Cytokeratin 8
9 Cytokeratin 18
10 Beta-actin
11 cytokeratin 19
12 Eukaryotic initiation factor 4A-I
13 14-3-3 protein epsilon
14 14-3-3 protein zetadelta (Protein kinase Cinhibitor protein-1)
15 Translationally controlled tumor protein (TCTP)
Among these autoantigens cytokeratin 8 cytokeratin 18 Fructose-bisphosphate aldolase and enolase were reacted with more than 60 of healthy individualsrsquo serum samples These antigens were intense spots in gels
Heat Shock proteins tubulins heterogeneous nuclear ribonucleoprotein actins cytokeratins 14-3-3 protein epsilon 14-3-3 protein zetadelta eukaryotic initiation factors Rho GDP-dissociation inhibitors phosphoglycerate mutase 1 G6PD GA3PD peroxiredoxins F-acting capping protein and triosephosphate isomerase were previously reported to induce humoral arm of the immune response in cancer
Translationally controlled tumor protein and electron transfer flavoprotein alpha-subunit have not been reported to induce humoral arm of the immune response in cancer so far
In terms of biological processes the identified proteins
belong to several groups
They are mainely involved in cell motility cytoskleton
stress response apoptosis cell signaling and
metabolism
Identification of other immunogenic proteins is
underway
Appel RD et al 1993 - SWISS-2DPAGE a database of two-dimensional gel electrophoresis images Electrophoresis 14 1232-1238
Appel RD Bairoch A Sanchez JC Vargas JR Golaz O Pasquali C and Hochstrasser DF 1996 ndash Federated two-dimensional electrophoresis database a simple means of publishing two-dimensional electrophoresis data Electrophoresis 17 540-546
Bjellqvist B Ek K Righetti PG Gianazza E Gorg A Westermeier R Postel W 1982 ndash Isoelectric focusing in immobilised pH gradients principle methodology and applications JBiochemBiophysMethods 6 317-339
Brazma A et al 2001 ndash Minimum information about a microarray experiment (MIAME)-towards standards for microarray data Nat genetics 29 365-71
Hoogland C Baujard Sanchez JC Hochstrasser DF and Appel RD 1997 ndash Make2ddb a simple package to set up a two-diensional electrophoresis database for the world wide web Electrophoresis 18 2755-2758
OFarrell 1975 - High resolution two-dimensional electrophoresis of proteins JBiolChem 25 250 4007-21
Time line ~ 4 days
bull Protein gt 250 kDa do not enter 2 SDS-PAGE properly
bull PH more than 9
bull PH less than 4
bull Hydrophobic proteins
bull Low abundant proteins
Immunoblotting Proteins from 2D gels were transferred to PVDFmembranes The membranes were blocked with 5 skimmed milk in PBS-Tween (PBS-T) for 3 h The membranes were incubated overnight at 4degC with individual sera diluted 1100 in PBS-T followed by secondary antibody (anti-human IgG conjugated with horseradish peroxidase) for 2 h with dilutions of 13000 Before and after addition of the secondary antibody membranes washed 4 times each time for 5 min in PBS-T To visualize antigenic components membranes were incubated in diaminobenzeden (DAB) and H2O2 for 15 min
List of protein spots that reacted with more than 30 of serum samples
and were identified by mass spectrometry
1Heat shock protein HSP 90-beta
2 precursor(GRP 78) 78 kDa glucose-regulated protein
3 Heat shock cognate 71 kDa protein
4 Stress-70 protein mitochondrial precursor
5 Tubulin beta-5 chain
6Tubulin alpha-6 chain and 60 kDa heat shock protein mitochondrial
precursor (Hsp60)
7 Heterogeneous nuclear ribonucleoprotein K (hnRNP K)
8 Cytokeratin 8
9 Cytokeratin 18
10 Beta-actin
11 cytokeratin 19
12 Eukaryotic initiation factor 4A-I
13 14-3-3 protein epsilon
14 14-3-3 protein zetadelta (Protein kinase Cinhibitor protein-1)
15 Translationally controlled tumor protein (TCTP)
Among these autoantigens cytokeratin 8 cytokeratin 18 Fructose-bisphosphate aldolase and enolase were reacted with more than 60 of healthy individualsrsquo serum samples These antigens were intense spots in gels
Heat Shock proteins tubulins heterogeneous nuclear ribonucleoprotein actins cytokeratins 14-3-3 protein epsilon 14-3-3 protein zetadelta eukaryotic initiation factors Rho GDP-dissociation inhibitors phosphoglycerate mutase 1 G6PD GA3PD peroxiredoxins F-acting capping protein and triosephosphate isomerase were previously reported to induce humoral arm of the immune response in cancer
Translationally controlled tumor protein and electron transfer flavoprotein alpha-subunit have not been reported to induce humoral arm of the immune response in cancer so far
In terms of biological processes the identified proteins
belong to several groups
They are mainely involved in cell motility cytoskleton
stress response apoptosis cell signaling and
metabolism
Identification of other immunogenic proteins is
underway
Appel RD et al 1993 - SWISS-2DPAGE a database of two-dimensional gel electrophoresis images Electrophoresis 14 1232-1238
Appel RD Bairoch A Sanchez JC Vargas JR Golaz O Pasquali C and Hochstrasser DF 1996 ndash Federated two-dimensional electrophoresis database a simple means of publishing two-dimensional electrophoresis data Electrophoresis 17 540-546
Bjellqvist B Ek K Righetti PG Gianazza E Gorg A Westermeier R Postel W 1982 ndash Isoelectric focusing in immobilised pH gradients principle methodology and applications JBiochemBiophysMethods 6 317-339
Brazma A et al 2001 ndash Minimum information about a microarray experiment (MIAME)-towards standards for microarray data Nat genetics 29 365-71
Hoogland C Baujard Sanchez JC Hochstrasser DF and Appel RD 1997 ndash Make2ddb a simple package to set up a two-diensional electrophoresis database for the world wide web Electrophoresis 18 2755-2758
OFarrell 1975 - High resolution two-dimensional electrophoresis of proteins JBiolChem 25 250 4007-21
Immunoblotting Proteins from 2D gels were transferred to PVDFmembranes The membranes were blocked with 5 skimmed milk in PBS-Tween (PBS-T) for 3 h The membranes were incubated overnight at 4degC with individual sera diluted 1100 in PBS-T followed by secondary antibody (anti-human IgG conjugated with horseradish peroxidase) for 2 h with dilutions of 13000 Before and after addition of the secondary antibody membranes washed 4 times each time for 5 min in PBS-T To visualize antigenic components membranes were incubated in diaminobenzeden (DAB) and H2O2 for 15 min
List of protein spots that reacted with more than 30 of serum samples
and were identified by mass spectrometry
1Heat shock protein HSP 90-beta
2 precursor(GRP 78) 78 kDa glucose-regulated protein
3 Heat shock cognate 71 kDa protein
4 Stress-70 protein mitochondrial precursor
5 Tubulin beta-5 chain
6Tubulin alpha-6 chain and 60 kDa heat shock protein mitochondrial
precursor (Hsp60)
7 Heterogeneous nuclear ribonucleoprotein K (hnRNP K)
8 Cytokeratin 8
9 Cytokeratin 18
10 Beta-actin
11 cytokeratin 19
12 Eukaryotic initiation factor 4A-I
13 14-3-3 protein epsilon
14 14-3-3 protein zetadelta (Protein kinase Cinhibitor protein-1)
15 Translationally controlled tumor protein (TCTP)
Among these autoantigens cytokeratin 8 cytokeratin 18 Fructose-bisphosphate aldolase and enolase were reacted with more than 60 of healthy individualsrsquo serum samples These antigens were intense spots in gels
Heat Shock proteins tubulins heterogeneous nuclear ribonucleoprotein actins cytokeratins 14-3-3 protein epsilon 14-3-3 protein zetadelta eukaryotic initiation factors Rho GDP-dissociation inhibitors phosphoglycerate mutase 1 G6PD GA3PD peroxiredoxins F-acting capping protein and triosephosphate isomerase were previously reported to induce humoral arm of the immune response in cancer
Translationally controlled tumor protein and electron transfer flavoprotein alpha-subunit have not been reported to induce humoral arm of the immune response in cancer so far
In terms of biological processes the identified proteins
belong to several groups
They are mainely involved in cell motility cytoskleton
stress response apoptosis cell signaling and
metabolism
Identification of other immunogenic proteins is
underway
Appel RD et al 1993 - SWISS-2DPAGE a database of two-dimensional gel electrophoresis images Electrophoresis 14 1232-1238
Appel RD Bairoch A Sanchez JC Vargas JR Golaz O Pasquali C and Hochstrasser DF 1996 ndash Federated two-dimensional electrophoresis database a simple means of publishing two-dimensional electrophoresis data Electrophoresis 17 540-546
Bjellqvist B Ek K Righetti PG Gianazza E Gorg A Westermeier R Postel W 1982 ndash Isoelectric focusing in immobilised pH gradients principle methodology and applications JBiochemBiophysMethods 6 317-339
Brazma A et al 2001 ndash Minimum information about a microarray experiment (MIAME)-towards standards for microarray data Nat genetics 29 365-71
Hoogland C Baujard Sanchez JC Hochstrasser DF and Appel RD 1997 ndash Make2ddb a simple package to set up a two-diensional electrophoresis database for the world wide web Electrophoresis 18 2755-2758
OFarrell 1975 - High resolution two-dimensional electrophoresis of proteins JBiolChem 25 250 4007-21
List of protein spots that reacted with more than 30 of serum samples
and were identified by mass spectrometry
1Heat shock protein HSP 90-beta
2 precursor(GRP 78) 78 kDa glucose-regulated protein
3 Heat shock cognate 71 kDa protein
4 Stress-70 protein mitochondrial precursor
5 Tubulin beta-5 chain
6Tubulin alpha-6 chain and 60 kDa heat shock protein mitochondrial
precursor (Hsp60)
7 Heterogeneous nuclear ribonucleoprotein K (hnRNP K)
8 Cytokeratin 8
9 Cytokeratin 18
10 Beta-actin
11 cytokeratin 19
12 Eukaryotic initiation factor 4A-I
13 14-3-3 protein epsilon
14 14-3-3 protein zetadelta (Protein kinase Cinhibitor protein-1)
15 Translationally controlled tumor protein (TCTP)
Among these autoantigens cytokeratin 8 cytokeratin 18 Fructose-bisphosphate aldolase and enolase were reacted with more than 60 of healthy individualsrsquo serum samples These antigens were intense spots in gels
Heat Shock proteins tubulins heterogeneous nuclear ribonucleoprotein actins cytokeratins 14-3-3 protein epsilon 14-3-3 protein zetadelta eukaryotic initiation factors Rho GDP-dissociation inhibitors phosphoglycerate mutase 1 G6PD GA3PD peroxiredoxins F-acting capping protein and triosephosphate isomerase were previously reported to induce humoral arm of the immune response in cancer
Translationally controlled tumor protein and electron transfer flavoprotein alpha-subunit have not been reported to induce humoral arm of the immune response in cancer so far
In terms of biological processes the identified proteins
belong to several groups
They are mainely involved in cell motility cytoskleton
stress response apoptosis cell signaling and
metabolism
Identification of other immunogenic proteins is
underway
Appel RD et al 1993 - SWISS-2DPAGE a database of two-dimensional gel electrophoresis images Electrophoresis 14 1232-1238
Appel RD Bairoch A Sanchez JC Vargas JR Golaz O Pasquali C and Hochstrasser DF 1996 ndash Federated two-dimensional electrophoresis database a simple means of publishing two-dimensional electrophoresis data Electrophoresis 17 540-546
Bjellqvist B Ek K Righetti PG Gianazza E Gorg A Westermeier R Postel W 1982 ndash Isoelectric focusing in immobilised pH gradients principle methodology and applications JBiochemBiophysMethods 6 317-339
Brazma A et al 2001 ndash Minimum information about a microarray experiment (MIAME)-towards standards for microarray data Nat genetics 29 365-71
Hoogland C Baujard Sanchez JC Hochstrasser DF and Appel RD 1997 ndash Make2ddb a simple package to set up a two-diensional electrophoresis database for the world wide web Electrophoresis 18 2755-2758
OFarrell 1975 - High resolution two-dimensional electrophoresis of proteins JBiolChem 25 250 4007-21
Among these autoantigens cytokeratin 8 cytokeratin 18 Fructose-bisphosphate aldolase and enolase were reacted with more than 60 of healthy individualsrsquo serum samples These antigens were intense spots in gels
Heat Shock proteins tubulins heterogeneous nuclear ribonucleoprotein actins cytokeratins 14-3-3 protein epsilon 14-3-3 protein zetadelta eukaryotic initiation factors Rho GDP-dissociation inhibitors phosphoglycerate mutase 1 G6PD GA3PD peroxiredoxins F-acting capping protein and triosephosphate isomerase were previously reported to induce humoral arm of the immune response in cancer
Translationally controlled tumor protein and electron transfer flavoprotein alpha-subunit have not been reported to induce humoral arm of the immune response in cancer so far
In terms of biological processes the identified proteins
belong to several groups
They are mainely involved in cell motility cytoskleton
stress response apoptosis cell signaling and
metabolism
Identification of other immunogenic proteins is
underway
Appel RD et al 1993 - SWISS-2DPAGE a database of two-dimensional gel electrophoresis images Electrophoresis 14 1232-1238
Appel RD Bairoch A Sanchez JC Vargas JR Golaz O Pasquali C and Hochstrasser DF 1996 ndash Federated two-dimensional electrophoresis database a simple means of publishing two-dimensional electrophoresis data Electrophoresis 17 540-546
Bjellqvist B Ek K Righetti PG Gianazza E Gorg A Westermeier R Postel W 1982 ndash Isoelectric focusing in immobilised pH gradients principle methodology and applications JBiochemBiophysMethods 6 317-339
Brazma A et al 2001 ndash Minimum information about a microarray experiment (MIAME)-towards standards for microarray data Nat genetics 29 365-71
Hoogland C Baujard Sanchez JC Hochstrasser DF and Appel RD 1997 ndash Make2ddb a simple package to set up a two-diensional electrophoresis database for the world wide web Electrophoresis 18 2755-2758
OFarrell 1975 - High resolution two-dimensional electrophoresis of proteins JBiolChem 25 250 4007-21
In terms of biological processes the identified proteins
belong to several groups
They are mainely involved in cell motility cytoskleton
stress response apoptosis cell signaling and
metabolism
Identification of other immunogenic proteins is
underway
Appel RD et al 1993 - SWISS-2DPAGE a database of two-dimensional gel electrophoresis images Electrophoresis 14 1232-1238
Appel RD Bairoch A Sanchez JC Vargas JR Golaz O Pasquali C and Hochstrasser DF 1996 ndash Federated two-dimensional electrophoresis database a simple means of publishing two-dimensional electrophoresis data Electrophoresis 17 540-546
Bjellqvist B Ek K Righetti PG Gianazza E Gorg A Westermeier R Postel W 1982 ndash Isoelectric focusing in immobilised pH gradients principle methodology and applications JBiochemBiophysMethods 6 317-339
Brazma A et al 2001 ndash Minimum information about a microarray experiment (MIAME)-towards standards for microarray data Nat genetics 29 365-71
Hoogland C Baujard Sanchez JC Hochstrasser DF and Appel RD 1997 ndash Make2ddb a simple package to set up a two-diensional electrophoresis database for the world wide web Electrophoresis 18 2755-2758
OFarrell 1975 - High resolution two-dimensional electrophoresis of proteins JBiolChem 25 250 4007-21
Appel RD et al 1993 - SWISS-2DPAGE a database of two-dimensional gel electrophoresis images Electrophoresis 14 1232-1238
Appel RD Bairoch A Sanchez JC Vargas JR Golaz O Pasquali C and Hochstrasser DF 1996 ndash Federated two-dimensional electrophoresis database a simple means of publishing two-dimensional electrophoresis data Electrophoresis 17 540-546
Bjellqvist B Ek K Righetti PG Gianazza E Gorg A Westermeier R Postel W 1982 ndash Isoelectric focusing in immobilised pH gradients principle methodology and applications JBiochemBiophysMethods 6 317-339
Brazma A et al 2001 ndash Minimum information about a microarray experiment (MIAME)-towards standards for microarray data Nat genetics 29 365-71
Hoogland C Baujard Sanchez JC Hochstrasser DF and Appel RD 1997 ndash Make2ddb a simple package to set up a two-diensional electrophoresis database for the world wide web Electrophoresis 18 2755-2758
OFarrell 1975 - High resolution two-dimensional electrophoresis of proteins JBiolChem 25 250 4007-21